U.S. patent number 6,413,768 [Application Number 09/204,117] was granted by the patent office on 2002-07-02 for expression plasmids.
This patent grant is currently assigned to University of Maryland. Invention is credited to James E. Galen.
United States Patent |
6,413,768 |
Galen |
July 2, 2002 |
Expression plasmids
Abstract
The present invention relates generally to a Plasmid Maintenance
System for the stabilization of expression plasmids encoding
foreign antigens, and methods for making and using the Plasmid
Maintenance System. The invention optimizes the maintenance of
expression plasmids at two independent levels by: (1) removing sole
dependence on balanced lethal maintenance systems; and (2)
incorporating a plasmid partition system to prevent random
segregation of expression vector plasmids, thereby enhancing their
inheritance and stability. The Plasmid Maintenance System may be
employed within a plasmid which has been recombinantly engineered
to express a variety of expression products.
Inventors: |
Galen; James E. (Owings Mills,
MD) |
Assignee: |
University of Maryland
(Baltimore, MD)
|
Family
ID: |
22756705 |
Appl.
No.: |
09/204,117 |
Filed: |
December 2, 1998 |
Current U.S.
Class: |
435/320.1;
530/300; 530/350; 530/403; 536/24.1 |
Current CPC
Class: |
C07K
14/25 (20130101); C07K 14/255 (20130101); C12N
1/20 (20130101); C12N 15/74 (20130101); A61K
2039/521 (20130101) |
Current International
Class: |
C07K
14/195 (20060101); C07K 14/25 (20060101); C07K
14/255 (20060101); C12N 1/20 (20060101); C12N
15/74 (20060101); C12N 015/63 () |
Field of
Search: |
;435/320.1 ;536/24.1
;530/300,350,403 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
Other References
Kenn Gerdes et al. Unique type of plasmid maintenance function:
postsegregational killing of plasmid-free cells vol. 83 pp.
3116-3120 May 1986.* .
Gerdes et al. Mechanism of post-segregational killing by the
hok/sok system of plasmid R1: sok antisense RNA regulates formation
of a hok mRNA species correlated with killing of plasmid-free cells
Molecular Microbiology 1990 4(11) 1818.* .
Jensen et al., "Programmed cell death in bacteria: proteic plasmid
stabilization system", Molecular Microbiology, 1995, 17 (2), pp.
205-210. .
Pecota et al., "Combining the hok/sok par DE, and pnd
Postsegregational Killer Loci To Enhance Plasmid Stability",
Applied and Environmental Microbiology, May 1997, 63, (5), pp.
1917-1924. .
Gerdes et al., "Effects of Genes Exerting Growth Inhibition and
Plasmid Stability on Plasmid Maintenance", Journal of Bacteriology,
Oct. 1987, 169 (10), pp. 4646-4650. .
Gerdes et al., "Antisense RNA-Regulated Programmed Cell Death",
Annu. Rev. Genet., 1997, pp. 1-31. .
Gultyaev et al., "Programmed Cell Death by hok/sok of Plasmid R1:
Coupled Nucleotide Covariations Reveal a Phylogenetically Conserved
Folding Pathway in the hok Family of mRNAs", J. Mol. Biol., 1997,
273, pp. 26-37. .
Franch et al., "Programmed Cell Death by hok/sok of Plasmid R-1:
Processing at the hok mRNA 3-end Triggers Structural Rearrangements
that Allow Translation and Antisense RNA Binding", J. Mol. Biol.,
1997, 273, pp. 38-51. .
Nikolaj Dam Mikkelsen and Kenn Gerdes, "Sok antisense RNA from
plasmid R1 is functionally inactivated by Rnase E and
polyadenylated by poly (A) polymerase I", Molecular Microbiology,
1997, 26 (2), pp. 311-320. .
Thomas Franch and Kenn Gerdes, "Programmed cell death in bacteria:
translational repression by mRNA end-pairing", Molecular
Microbiology, 1996, 21 (5), pp. 1049-1060. .
Kenn Gerdes and Soren Molin, "Partitioning of Plasmid R1 Structural
and Functional Analysis of the par A Locus", J. Mol. Biol., 1986,
190, pp. 269-279. .
Gerdes et al., "Plasmid Stabilization by Post-Segregational
Killing", Genetic Engineering, 1997, 19, pp. 49-61. .
Gerdes et al., "Stable Inheritance of Plasmid R1 Requires Two
Different Loci", Journal of Bacteriology, Jan. 1985, 161 (1), pp.
292-298. .
Kuowei Wu and Thomas K. Wood, "Evaluation of the hok/sok Killer
Locus for Enhanced Plasmid Stability", Biotechnology and
Bioengineering, 1994, 44, pp. 912-921. .
Thomas K. Wood and Steven W. Peretti, "Effect of Chemicall-Induced,
Cloned-Gene Expression on Protein Synthesis in E. coli",
Biotechnology and Engineering, 1991, 38, pp. 397-412. .
Kenn Gerdes, "The PARB (Hok/Sok) Locus of Plasmid R1: A General
Purpose Plasmid Stabilization System", Biotechnology, Dec. 1998, 6,
pp. 1402-1405. .
Bravo et al., ."Identification of components of a new stability
system of plasmid R1, ParD, that is close to the origin or
replication of this plasmid", Mol Gen Genet, 1987, pp. 101-110.
.
Ruiz-Echevarria et al., "The kis genes of the parD maintenance
system of plasmid R1 form an operon that is autoregulated at the
level of transcription by the co-ordinated action of the Kis and
Kid proteins", Molecular Microbiology, 1997 5 (11), pp. 2685-2693.
.
Ruiz-Echevarria et al., "Translational coupling and limited
degradation of a polycistronic messenger modulate differential gene
expression in the parD stability system of plasmid R1", Gen. Genet,
1995, 248, pp. 599-609. .
Ruiz-Echevarria et al., "Kid, a Small Protein of the parD Stability
System of Plasmid R1, is an Inhibitor of DNA Replication Acting at
the Initiation of DNA Synthesis", J. Mol. Biol., 1995, 247, pp. 568
571. .
Bravo et al., "Killing of Escherichia coli cells modulated by
components of the stability system ParD of plasmid R1", Mol Gen
Genet, 1988, 215, pp. 146-151. .
Nordstrom et al., "Control of Replication of Bacterial Plasmids:
Genetics, Molecular Biology, and Physiology of the Plasmid R1
System", Academic Press Inc., 1984, pp. 71-91. .
Kurt Nordstrom and E. Gerhart H. Wagner, "Kinetic aspects of
control of plasmid replication by antisense RNA", Biosci, Jul.
1994, pp. 294-300. .
Kim Pedersen and Kenn Gerdes, "Multiple hok genes on the chromosome
of Escherichia coli", Molecular Microbiology, 1999, 32 (50), pp.
1090-1102. .
J. Dolfing et al., "Proposed New Nomenclature for SLT (VT) Family",
ASM News, 62, (3), 1996, pp. 117-119. .
Takeda et al.; "Bacterial Toxins and Virulence Factors in Disease",
Handbook of Natural Toxins, 8, pp. 313-327, 1995. .
Acheson et al., "Nomenclature of enterotoxins", The Lancet, 351,
Apr. 4, 1998, pp. 1003. .
Yaeta ENDO et al., "Site of action of a Vero toxin (VT2) from
Escherichia coli O157:H7 and of Shiga toxin on eukaryotic ribosomes
RNA N-glycosidase activity of the toxins", Eur. J. Biochem, 1988,
171, pp. 45-50. .
Gerdes et al., "Unique type of plasmid maintenance function:
Postsegragational killing of plasmid-free cells", Proc. Natl. Acad.
Sci. USA, May 1986, 83, pp. 3116-3120. .
Carlton Gyles, "Escherichia coli cytotoxins and enterotoxins", Can.
J. Microbiol., 1992, 38, pp. 7323-746. .
Matthew P. Jackson et al., "Nucleotide Sequence analysis and
comparison of the structural genes for Shiga-like toxin I and
Shiga-like toxin II encoded by bacteriophages from Escherichia coli
933", Federation of European Microbiological Societies, 1987, 44,
pp. 109-114. .
Vernon L. Tesh et al., "Comparison of the Relative Toxicities of
Shiga-Like Toxins Type I and Type II for Mice", Infection and
Immunity, Aug. 1993, 61 (8), pp. 3392-3402. .
Susanne W. Lindgren et al., The Specific Activities of Shiga-Like
Toxin Type II (SLT-11) and SLT-II-Related Toxins of
Enterohemorrhagic Escherichia coli Differ When Measured by Vero
Cell Cytotoxicity but Not by Mouse Lethality, Infection and
Immunity, Feb. 1994, 62 (2), pp. 623-631. .
Lawrence M. Sung et al., "Transcription of the Shiga-Like Toxin
Type II and Shiga-Like Toxin II Variant Operons of Escherichia
coli", Journal of Bacteriology, Nov. 1990, 172 (11), pp. 6386-6395.
.
Inge Muhldorfer et al., "Regulation of the Shiga-Like Toxin II
Operon in Escherichia coli", Infection and Immunity, Feb. 1996, 64
(2), pp. 495-502. .
Clare K. Schmitt, "Two Copies of Shiga-Like Toxin II-Related Genes
Common in Enterohemorrhagic Escherichia coli Strains Are
Responsible for the Antigenic Heterogeneity of the O157:H-Strain
E32511", Infection and Immunity, Mar. 1991, 59 (3), pp. 1065-1073.
.
Debra L. Weinstein et al., "Cloning and Sequencing of a Shiga-Like
Type II Variant from an Escherichia coli Strain Responsible for
Edema Disease of Swine", Journal of Bacteriology, Sep. 1988, 170
(9), pp. 4223-1230. .
C.L. Gyles et al., "Cloning and nucleotide sequence analysis of the
genes determining verocytotoxin production in a porcine edema
disease isolate of Escherichia coli", Microbial Pathologies, 1988,
5, pp. 419-426. .
Adrienne W. Paton et al., "Comparative Toxicity and Virulence of
Escherichia coli Clones Expressing Variant and Chimeric Shiga-Like
Toxin Type II Operons", Infection and Immunity, Jul. 1995, 63 (7),
pp. 2450-2458. .
Adrienne W. Paton et al., "Polymerase chain reaction amplification,
cloning and sequencing of variant Escherichia coli Shiga-like toxin
type II operons", Microbial Pathogenesis, 1993, 15, pp. 77-82.
.
Hideaki Ito et al., "Cloning and nucleotide sequencing of Vero
toxin 2 variant genes from Escherichia coli 091:H21 isolated from a
patient with the hemoloytic uremic syndrome", Microbial
Pathogenesis, 1990, 8, pp. 47-60. .
Marie E. Fraser et al., "Crystal structure of the holotoxin from
Shigella dysenteriae at 2.5 A resolution", Structural Biology, Jan.
1994, 1 (1), pp. 59-64. .
Penolope E. Stein et al., "Crystal structure of the cell-binding B
oligomer of verotoxin-1 from E. coli", Nature, Feb. 1992, 355, pp.
748-750. .
Per-Georg et al., "Modelling of the interaction of verotoxin-1
(VT1) with its glycolipid receptor, globotriasylceramide
(GB.sub.3)", Int. J. Biol. Macromol., 1995, 17 (3-4), pp. 199-204.
.
Per-Georg et al., "Two distinct binding sites for globotriaosyl
ceramide on verotoxins: identification by molecular modelling and
confirmation using deoxy analogues and a new glycolipid receptor
for all verotoxins", Chemistry & Biology, 1996, 3 (4), pp.
263-275. .
Hong Ling et al., "Structure of the Shiga-like Toxin I B-Pentamer
Complexed with an Analogue of Its Receptor GB.sub.3 ", Bichemistry,
1998, 37, pp. 1777-1788. .
Carolyn J. Hovde et al., "Evidence that glutamic acid 167 is an
active-site residue of Shiga-like toxin I", Proc. Natl. Acad. Sci.
USA, Apr. 1988, 85, pp. 2568-2572. .
Shinji Yamasaki et al., "importance of arginine at position 170 of
the A subunit of Vero toxin 1 produced by enterohemorrhagic
Escherichia coli for toxin activity", Microbial Pathogenesis, 1991,
11, pp. 1-9. .
Matthew P. Jackson et al., "Mutational Analysis of the Shiga Toxin
and Shiga-Like Toxin II Enzymatic Subunits", Journal of
Bacteriology, Jun. 1990, 172 (6), pp. 3346-3350. .
V.M. Gordon et al., "An Enzymatic Mutant of shiga-Like Toxin II
Variant Is a Vaccine Candidate for Edema Disease of Swine",
Infection and Immunity, Feb. 1992, 60 (2), pp. 485-490. .
B.T. Bosworth et al., "Vaccination with Genetically Modified
Shiga-Like Toxin Iie Prevents Edema Disease in Swine", Infection
and Immunity, Jan. 1996, 64 (1), pp. 55-60. .
Matthew P. Jackson, "Functional Analysis of the Shiga Toxin and
Shiga-Like Toxin Type II Variant binding Subunits by Using
Site-Directed Mutagenesis", Journal of Bacteriology, Feb. 1990, 172
(20), pp. 653-658. .
Clifford Clark, "Phenylalanine 30 plays an important role in
receptor binding of verotoxin-1", Molecular Microbiology, 1996, 19
(4), pp. 891-899. .
Darrin J. Bast, "Toxicity and Immunogenicity of a Verotoxin 1
Mutant with Reduced Globotriaosylceramide Receptor Binding in
Rabbits", Infection and Immunity, Jun. 1997, 65 (6), pp. 2019-2028.
.
L.P. Perera et al, "Identification of Three Amino Acid Residues in
the B Subunit of Shiga Toxin and Shiga-Like Toxin Type II That are
Essential for Holotoxin Activity", Journal of Bacteriology, Feb.
1991, 173 (3), pp. 1151-1160. .
L.P. Perera et al., "Mapping the Minimal Contiguous Gene Segment
That Encodes Functionally Active Shiga-Like Toxin II", Infection
and Immunity, Mar. 1991, 59 (3), pp. 829-835. .
Frances Pouch Downes et al., "Affinity Purification and
Characterization of Shiga-Like Toxin II and Production of
Toxin-Specific Monoclonal Antibodies", Infection and Immunity, Aug.
1988, 56 (8), pp. 1926-1933. .
Guo-Fu Su et al., "Construction of Stable LamB-shiga Toxin B
Subunit Hybrids: Analysis of Expression in Salmonella typhimurium
aroA Strains and Stimulation of B Subunit-Specific Mucosal and
Serum Antibody Responses", Infection and Immunity, Aug. 1992, 60
(8), pp. 3345-3359. .
Gue-fu Su et al., "Extracellular export of Shiga Toxin
B-subunit/haemolysin A (C-terminus) fusion protein expressed in
Salmonella typhimurium aro A-mutant and stimulation of B-subunit
antibody responses in mice", Microbial Pathogenesis, 1992, 13, pp.
465-476. .
Susan E. Richardson et al., "Experimental Verocytotoxemia in
Rabbits", Infection and Immunity, Oct. 1992, 60 (10), pp.
4154-4167. .
S. Nelson et al., "Biological activity of verocytotoxin (VT)2c and
VT1/VT2c chimeras in the rabbit model", Elsevier Science, 1994, pp.
245-249. .
M. Bielaszewska et al., "Localization of Intravenously Administered
Verocytotoxins (Shiga-Like Toxins) 1 and 2 in Rabbits Immunized
with Homologous and Heterologous Toxoids and Toxin Subunits",
Infection and Immunity, Jul. 1997, 65 (7), pp. 2509-2516. .
Stephen J. Streatfield et al., "Intermolecular interactions between
the A and B subunits of heat-labile enterotoxin from Escherichia
coli promote holotoxin assembly and stability in vivo", Proc. Natl.
Acad. Sci. USA, Dec. 1992, 89, pp. 12140-12144. .
David W.K. Acheson et al., "Protective Immunity to Shiga-Like Toxin
I following Oral Immunization with Shiga-Like Toxin I
B-Subunit-Producing Vibrio cholerae CVD 103-HgR", Infection and
Immunity, Jan. 1996, 64 (1), pp. 355-357. .
Elizabeth A. Wadolkowski et al., "Acute Renal Tubular Necrosis and
Death of Mice Orally Infected with Escherichia coli Strains That
Produce Shiga-Like Toxin Type II", Infection and Immunity, Dec.
1990, 58 (12), pp. 3959-3965. .
Diana Karpman et al, "The Role of Lipopolysaccharide and Shiga-like
Toxin in a Mouse Model of Escherichia coli 0157:H7 Infection", The
Journal of Infectious Diseases, 1997 175, pp. 611-620. .
Chandra B. Louise and Tom G. Obrig, "Specific Interaction of
Escherichia coli 0157:H7-Derived Shiga-like Toxin II with Human
Renal Endothelial Cells", The Journal of Infectious Diseases, 1995,
172, pp. 1397-1401. .
Beth Boyd and Clifford Lingwood, "Verotoxin Receptor Glycolipid in
Human Renal Tissue", Nephron, 1989, pp. 207-210. .
Carla Zoja et al., "Verotoxin glycolipid receptors determine the
localization of microangiopathic process in rabbits given
verotoxin-1", J Lab Clin Med., Aug. 1992, 120 (2), pp. 229-238.
.
J. Yu and J.B. Kaper, "Cloning and characterization of the eae gene
of enterohaemorrhagic Escherichia coli 0157:H7", Molecular
Microbiology, 1992 6 (3), pp. 411-417. .
Timothy K. McDaniel et al., "A genetic locus of enterocyte
effacement conserved among diverse enterobacterial pathogens",
Proc. Natl. Acad. Sci. USA, Feb. 1995, 932, pp. 1664-1668. .
Karen G. Jarvis et al., "Enteropathogenic Escherichia coli contains
a putative type III secretion system necessary for the export of
proteins involved in attaching and effacing lesion formation",
Proc. Natl. Acad. Sci. USA, Aug. 1995, 92, pp. 7996-8000. .
Karen G. Jarvis et al., "Secretion of Extracellular Proteins by
Enterohemorrhagic Escherichia coli via a Putative Type III
Secretion System", Infection and Immunity, Nov. 1996, 64 (11), pp.
4826-4829. .
Titia K. Sixma et al., "Comparison of the B-Pentamers of
Heat-Labile Enterotoxin and Verotoxin-1: Two Structures with
Remarkable Similarity and Dissimilarity", Bichemistry, 1993, 32,
pp. 191-198. .
Marianne Mangeney et al., "Apoptosis Induced in Burkitt's Lymphoma
Cells via Gb3/CD77, a Glycolipid Antigen.sup.1 ", Cancer Research,
Nov. 1, 1993, 53, pp. 5314-5319. .
Sylvia Franke et al., "Clonal Relatedness of Shiga-Like
Toxin-Producing Escherichia coli O101 Strains of Human and Porcine
Origin", Journal of Clinical Microbiology, Dec. 1995, 33 (12), pp.
3174-3178. .
San-Hyun Kim et al., "Cloning and Sequence Analysis of Another
Shiga-Like Toxin 11e Variant Gene (slt-llera) from an Escherichia
coli R107 Strain Isonlated from Rabbit", Microbiol. Immunol., 1997,
41 (10), pp. 805-808. .
Joan R. Butterton et al., "Coexpression of the B Subunit of Shiga
Toxin 1 and EaeA from Enterohemorrhagic Escherichia coli in Vibrio
cholerae Vaccine Strains", Isfection and Immunity, Jun. 1997, 65
(6), pp. 2127-2135. .
Nataro and Kaper, "Enterohemorrhagic E. coli" and "Diarrheagenic E.
Coli", American Society for Microbiology, 1998, 11, pp. 164-178.
.
Samir Taga et al., "Intracellular Signaling Events in CD77-Mediated
Apoptosis of Burkitt S Lymphoma Cells", Blood, Oct. 1, 1997, 90
(7), pp. 2757-2767. .
Judette E. Haddad et al., "Minimum Domain of the Shiga Toxin A
Subunit Required for Enzymatic Activity", Journal of Bacteriology,
Aug. 1993, 175 (16), pp. 4970-4978. .
Victor P. J. Gannon et al., "Molecular cloning and nucleotide
sequence of another variant of the Escherichia coli Shiga-like
toxin II family", Journal of General Microbiology, 1990, 136, pp.
1125-1135. .
A.D. O'Brien et al., "Shiga Toxin: Biochemistry, Genetics, Mode of
Action, and Role in Pathogenesis", Microbiology and Immunology,
1992, 180, pp. 66-93. .
William Montfort et al., "The Three-dimensional Structure of Ricin
at 2.8 A", The Journal of Biological Chemistry, Apr. 15, 1987, 262
(11), pp. 5398-5403. .
Susanne W. Lindgren et al., "Virulence of Enterohemorrhagic
Escherichia coli O91:H21 Clinical Isolates in an Orally Infected
Mouse Model", Infection and Immunity, Sep. 1993, 61 (9), pp.
3832-3842. .
Rasmussen, P.B., et al., "Genetic analysis of the parB+ locus of
Plasmid R1", Mol. Gen. Genet., 1987, 209 (1), pp. 122-128. .
Gerdes, K., et al., "Mechanism of post-segregational killing by the
hok/sok system of plasmid R1:sok antisense RNA regulates formation
of hok mRNA species correlated with killing of plasmid-free cells",
Mol. Microbiol., 1990, 4 (11), pp. 1807-1818. .
"Custom DNA/RNA Synthesis, Order Form", Fisher Scientific, DNA Name
5SHOK-TET2 Form No. 070697. .
"Custom DNA/RNA Synthesis, Order Form", Fisher Scientific, DNA Name
3SHOK-TET2, Form No. 070967. .
"Custom DNA/RNA Synthesis, Order Form", Fisher Scientific, DNA Name
3PDC-TET2, Form No. 070697. .
"Custom DNA/RNA Synthesis, Order Form", Fisher Scientific, DNA Name
5PDC-TET2, Form No. 070697. .
Thomas Thisted et al., "Mechanism of Post-segregational Killing:
Secondary Structure Analysis of the Entire Hok mRNA from Plasmid R1
Suggests a Folds-back Structure that Prevents Translation and
Antisense RNA Binding", J. Mol. Biol., 1995, pp. 859-873. .
Thomas Thisted et al., "Mechanism of post-segregational killing:
translation of Hok, srnB and Pnd mRNAs of plasmids R1, F and R483
is activated by 3'-end processing", The EMBO Journal, 1994, 13 (8),
1950-1959. .
Summers, D.K., The Biology of Plasmids, 1996, pp. 65-91..
|
Primary Examiner: Yucel; Remy
Attorney, Agent or Firm: Knobbe, Martens, Olson & Bear
LLP
Government Interests
The development of the present invention was supported by the
University of Maryland, Baltimore, Md. and by funding from the
National Institutes of Health under contract number NIH
R01-Al29471. The United States Government has a non-exclusive,
irrevocable, paid-up license to practice or have practiced for or
on behalf of the Unites States the invention herein as provided for
by the terms of the above mentioned contracts awarded by the United
States Government.
Claims
What is claimed is:
1. An expression plasmid comprising:
a restricted-copy-number origin of replication, wherein the
expression plasmid is limited to a copy number from about 2 to 75
copies per cell and wherein the origin of replication has
transcriptional terminators at the 5' and 3' end of said origin of
replication such that said origin of replication is isolated from
transcription beginning outside of the origin of replication;
a selection marker;
a post-segregational killing system; and
an inducible promoter linked to a heterologous antigen coding
sequence, wherein the inducible promoter is an ompC promoter (SEQ
ID NO: 1, from 7 to 464).
2. The expression plasmid of claim 1, wherein the origin of
replication is selected from the group consisting oriE1 (SEQ ID NO:
1 (from 1251 to 1932)), ori15A (SEQ ID NO: 2 (from 1251 to 1899)),
or ori101 (SEQ ID NO: 3 (from 1251 to 3196)).
3. The expression plasmid of claim 1, wherein the transcriptional
terminator comprises SEQ ID NO:1 (from 1954 to 2374).
4. The expression plasmid of claim 1, wherein the transcriptional
terminator comprises SEQ ID NO:1 (from 1215 to 1251).
5. The expression plasmid of claim 1, wherein the selection marker
is the tetA resistance gene (SEQ ID NO:1 from 3591 to 2403).
6. The expression plasmid of claim 1, wherein the
post-segregational killing system consists of hok-sok (SEQ ID NO:1
(from 3612 to 4196)).
7. The expression plasmid of claim 1, wherein the plasmid comprises
SEQ ID NO: 1.
8. The expression plasmid of claim 1, wherein the heterologous
antigen coding sequence encodes a detoxified Shiga toxin comprising
a mutated segment of amino acids comprising SEQ ID NO: 4.
9. The expression plasmid of claim 1, wherein the heterologous
antigen coding sequence encodes a detoxified Shiga toxin comprising
a mutated segment of amino acids comprising SEQ ID NO: 5.
10. The expression plasmid of claim 1, wherein the heterologous
antigen coding sequence encodes a detoxified Shiga toxin comprising
a mutated segment of amino acids comprising SEQ ID NO: 6.
11. The expression plasmid of claim 1, wherein the heterologous
antigen coding sequence encodes a detoxified Shiga toxin comprising
a mutated segment of amino acids comprising SEQ ID NO: 7.
12. The expression plasmid of claim 1, further comprising a
partitioning system.
13. The expression plasmid of claim 1, wherein the partitioning
system comprises par (SEQ ID NO: 3 (from 2824 to 3196).
Description
TABLE OF CONTENTS
1. BACKGROUND OF THE INVENTION
1.1 Field of the Invention
1.2 Description of Related Art
1.2.1 Bacterial Live Vector Vaccines
1.2.2 Attenuated Salmonella typhi as a live vector strain
1.2.3 Plasmid Instability
1.2.4 Plasmid Stabilization Systems
1.2.5 Antibiotic Resistance
1.2.6 Segregational Plasmid Maintenance Functions
1.2.7 Post-Segregational Killing (PSK) Functions
1.2.7.1 Proteic Maintenance System: The phd/doc System
1.2.7.2 Antisense Maintenance System: The hok-sok System
1.2.7.3 Balanced Lethal Systems
2. SUMMARY OF THE INVENTION
3. DEFINITIONS
4. BRIEF DESCRIPTION OF THE DRAWINGS
5. DETAILED DESCRIPTION OF THE INVENTION
5.1 Suicide Vectors
5.2 Plasmid-based Expression of Heterologous Antigens
5.3 Balanced Lethal Systems
5.4 Segregation Limitations
5.5 Catalytic Activity Limitations
5.6 The Non-Catalytic SSB PSK Function
5.7 Expression Plasmids and Self-Contained Genetic Cassettes
5.8 Components of the Antigen Expression and Replication
Cassette
5.8.1 Promoter
5.8.2 Origin of Replication
5.8.3 Expressed Protein or Peptide
5.8.4 Heterologous Antigens
5.8.4.1 The Shiga Toxin Family
5.8.5 Site-Specific Mutagensis of Shiga Toxins
5.9 Pharmaceutical Formulations
6. EXAMPLES
6.1 pGen Structure
6.2 P.sub.OmpC Promoter
6.3 Modified OmpC Promoter
6.4 Origins of Replication and Selection Cassettes
6.5 The Hok-Sok Antisense Post-Segregational Killing Locus
6.6 Complementation-Based Killing System
6.7 Conclusions
7. REFERENCES
THE CLAIMS
ABSTRACT OF THE DISCLOSURE
1. BACKGROUND OF THE INVENTION
1.1 Field of the Invention
The present invention relates generally to expression plasmids
stabilized by a Plasmid Maintenance System (as defined herein)
capable of expressing a protein or peptide, such as an antigen for
use in a live vector vaccine, and methods for making and using the
stabilized plasmids. The invention optimizes the maintenance of
expression plasmids at two independent levels by: (1) removing sole
dependence on catalytic balanced lethal maintenance systems; and
(2) incorporating a plasmid partition system to prevent random
segregation of expression plasmids, thereby enhancing inheritance
and stability.
1.2 Description of Related Art
Set forth below is a discussion of art relevant to the present
invention.
1.2.1 Bacterial Live Vector Vaccines
Bacterial live vector vaccines represent an important and promising
strategy in the field of vaccine development. These vaccines
deliver antigens to a host immune system by expressing the antigens
from genetic material contained within a bacterial live vector. The
genetic material is typically a replicon, such as a plasmid. The
antigens may include a wide variety of proteins and/or peptides of
bacterial, viral, parasitic or other origin.
Among the bacterial live vectors currently under investigation are
attenuated enteric pathogens (e.g., Salmonella typhi, Shigella,
Vibrio cholerae), commensals (e.g., Lactobacillus, Streptococcus
gordonii) and licensed vaccine strains (e.g., BCG). For the reasons
discussed below, S. typhi is a particularly attractive candidate
for human vaccination.
1.2.2 Attenuated Salmonella typhi as a Live Vector Strain
S. typhi is a well-tolerated live vector that can deliver multiple
unrelated immunogenic antigens to the human immune system. S. typhi
live vectors have been shown to elicit antibodies and a cellular
immune response to expressed antigen. Examples of antigens
successfully delivered by S. typhi include the non-toxigenic yet
highly immunogenic fragment C of tetanus toxin and the malaria
circumsporozoite protein from Plasmodium falciparum.
S. typhi is characterized by enteric routes of infection, a quality
which can enable oral vaccine delivery. S. typhi also infects
monocytes and macrophages and can therefore target antigens to
professional APCs.
Expression of an antigen by S. typhi generally requires
incorporation of a recombinant plasmid encoding the antigen.
Consequently, plasmid stability is a key factor in the development
of high quality attenuated vaccines with the ability to
consistently express foreign antigens.
Attenuated S. typhi vaccine candidates for use in humans should
possess at least two well separated and well defined mutations that
independently cause attenuation, since the chance of in vitro
reversion of such double mutants would be negligible.
The attenuated vaccine candidate S. typhi CVD908 possesses such
properties. CVD908 contains two non-reverting deletion mutations
within the aroC and aroD genes. These two genes encode enzymes
critical in the biosynthetic pathway leading to synthesis of
chorismate, the key precursor required for synthesis of the
aromatic amino acids phenylalanine, tyrosine, and tryptophan.
Chorismate is also required for the synthesis of p-aminobenzoic
acid; after its conversion to tetrahydrofolate, p-aminobenzoic acid
is converted to the purine nucleotides ATP and GTP.
1.2.3 Plasmid Instability
Plasmidless bacterial cells tend to accumulate more rapidly than
cells containing active plasmids. Summers, The Biology of Plasmids,
65-91, 1996 (incorporated herein by reference). One reason for this
increased rate of accumulation is that the transcription and
translation of plasmid genes imposes a metabolic burden which slows
cell growth and gives plasmidless cells a competitive advantage.
Furthermore, foreign plasmid gene products are sometimes toxic to
the host cell.
Stable inheritance of plasmids is desirable in the field of
attenuated bacterial live vector vaccines to ensure successful
continued antigen production, as well as in commercial bioreactor
operations in order to prevent bioreactor takeover by plasmidless
cells.
Stable inheritance of a plasmid generally requires that: (1) the
plasmid must replicate once each generation, (2) copy number
deviations must be corrected, and (3) upon cell division, the
products of replication must be distributed to both daughter cells.
Summers, The Biology of Plasmids, 65-91, 1996 (the entire
disclosure of which is incorporated herein by reference).
Although chromosomal integration of foreign genes confers stability
to such sequences, the genetic manipulations involved can be
difficult, and the drop in copy number of the heterologous gene
often results in production of insufficient levels of heterologous
antigen to ensure an optimal immune response. Introduction of
heterologous genes onto multicopy plasmids maintained within a live
vector strain is a natural solution to the copy number problem.
Genetic manipulation of such plasmids for controlled expression of
such heterologous genes is straightforward; however, resulting
plasmids can become unstable in vivo, resulting in loss of these
foreign genes.
1.2.4 Plasmid Stabilization Systems
In nature bacterial plasmids are often stably maintained. See
Gerdes et al. Annu. Rev. Genet., 31:1-31, 1997 (incorporated herein
by reference). In some circumstances, stable maintenance may simply
result from a high copy number. However, many proteins, such as
antigens, which may be desirably produced by bacterial cells are
toxic if produced in large amounts per cell. Therefore, it is
desirable to provide stable lower copy number plasmids for use in
bacterial cells.
Stable inheritance of naturally occurring lower copy number
plasmids can depend on the presence of certain genetic systems
which actively prevent the appearance of plasmid-free progeny. A
recent review of plasmid stabilization systems can be found in
Jensen et al. Molecular Microbiol. 17:205-210, 1995 (incorporated
herein by reference).
1.2.5 Antibiotic Resistance
One means for stabilizing plasmids is to provide an antibiotic
resistance gene on the plasmid and to grow the cells in
antibiotic-enriched media. However, this method is subject to a
number of difficulties. The antibiotic resistance approach is
expensive, requiring the use of costly antibiotics and perhaps,
more importantly, the use of antibiotics in conjunction with in
vivo administration of vaccine vectors may promote the growth of
antibiotic-resistant bacteria and is currently forbidden by the
U.S. Food and Drug Administration.
In large-scale production applications, the use of antibiotics may
impose other limitations. With respect to commercial bioreactors,
antibiotic resistance mechanisms can degrade the antibiotic and
permit a substantial population of plasmidless cells to persist in
the culture. Such plasmidless cells are unproductive and decrease
the output of the bioreactor.
There is therefore a need in the art for a plasmid stabilization
system specifically designed for use in bacterial live vector
vaccines which does not rely on antibiotic resistance, and
preferably which is also useful in commercial bioreactor
applications.
1.2.6 Segregational Plasmid Maintenance Functions
Stable lower copy number plasmids typically employ a partitioning
function that actively distributes plasmid copies between daughter
cells. Exemplary partitioning functions include, without
limitation, systems of pSC101, the F factor, the P1 prophage, and
IncFII drug resistance plasmids. Such functions are referred to
herein as "SEG" functions.
1.2.7 Post-Segregational Killing (PSK) Functions
PSK plasmid maintenance functions typically employ a two component
toxin-antitoxin system and generally operate as follows: The
plasmid encodes both a toxin and an antitoxin. The antitoxins are
less stable than the toxins, which tend to be quite stable. In a
plasmidless daughter cell, the toxins and anti-toxins are no longer
being produced; however, the less stable antitoxins quickly
degrade, thereby freeing the toxin to kill the cell.
The toxins are generally small proteins and and the antitoxins are
either small proteins (proteic systems such as phd/doc) or
antisense RNAs which bind to the toxin-encoding mRNAs preventing
their synthesis (antisense systems such as hok/sok).
Balanced lethal systems discussed below in Section 1.2.6.3 are an
example of an artificial PSK function.
1.2.7.1 Proteic Maintenance System: The phd/doc System
In proteic PSK functions, both the toxin and antitoxin are
synthesized from operons in which the gene encoding the antitoxin
is upstream of the gene encoding the toxin. These operons
autoregulate transcription levels, and synthesis of the encoded
proteins is translationally coupled. The antitoxin is generally
synthesized in excess to ensure that toxin action is blocked. The
unstable antitoxins are constantly degraded by host-encoded
proteases, requiring constant synthesis of antitoxin to protect the
cell. Upon loss of the plasmid, antitoxins are no longer produced
and the existing antitoxins rapidly degrade. This frees the toxin
to kill the host cell.
The phd/doc system is an example of a proteic PSK function. The
phd/doc system occurs naturally within the temperate bacteriophage
P1, which lysogenizes Escherichia coli as an .about.100 kb plasmid.
This maintenance locus encodes two small proteins: the toxic 126
amino acid Doc protein causes death on curing of the plasmid by an
unknown mechanism, and the 73 amino acid Phd antitoxin prevents
host death, presumably by binding to and blocking the action of
Doc.
Phd and Doc are encoded by a single transcript in which the ATG
start codon of the downstream doc gene overlaps by one base the TGA
stop codon of the upstream phd gene. Expression of these two
proteins is therefore translationally coupled, with Phd synthesis
exceeding synthesis of the toxic Doc protein.
In addition, transcription of this operon is autoregulated at the
level of transcription through the binding of a Phd-Doc protein
complex to a site which blocks access of RNA polymerase to the
promoter of the operon as concentrations of both proteins reach a
critical level. Although Doc appears to be relatively resistant to
proteolytic attack, Phd is highly susceptible to cleavage. The PSK
mechanism of a plasmid-encoded phd-doc locus is therefore activated
when bacteria spontaneously lose this resident plasmid, leading to
degradation of the Phd antitoxin and subsequent activation of the
Doc toxin which causes cell death.
1.2.7.2 Antisense Maintenance System: The hok-sok System
In antisense maintenance systems, the antitoxins are antisense RNAs
that inhibit translation of toxin-encoding mRNAs. Like the
antitoxin peptides, the antisense RNAs are less stable than the
toxin-encoding mRNA. Loss of the plasmid permits existing
antitoxins to degrade, thereby permitting synthesis of the toxin
which kills the host cell.
An example of an antisense maintenance system is the hok-sok
system, encoded by the parB locus of plasmid R1. See Thisted et
al., J. Mol. Biol. 247:859-873, 1995 (incorporated herein by
reference). The system is comprised of three genes: hok, sok and
mok.
Hok is a membrane-associated protein which irreversibly damages the
cell membrane, killing host cells. Expression of Hoc from hok mRNA
leads to a loss of cell membrane potential, arrest of respiration,
changes in cell morphology, and death.
The sok gene encodes a trans-acting RNA which blocks translation of
hok mRNA, thereby preventing Hoc killing of host cells. The sok RNA
is less stable than hok mRNA and is expressed from a relatively
weak promoter. Gerdes et al. Annu. Rev. Genet., 31:1-31, 1997. The
mechanism by which sok RNA blocks translation of Hok in
plasmid-containing cells became apparent only after the
identification of mok (modulation of killing), a third gene in the
parB locus. The mok open reading frame overlaps with hok, and is
necessary for expression and regulation of hok translation.
The sok antisense RNA forms a duplex with the 5' end of the mok-hok
message rendering the mok ribosome binding site inaccessible to
ribosomes and promoting RNase III cleavage and degradation of the
mRNA. In the absence of mok translation, hok is not expressed from
intact message, even though its own ribosome binding site is not
directly obscured by sok RNA.
When a plasmid-free cell is formed, the unstable sok RNA decays
much more rapidly than the stable mok-hok message. When the
protection afforded by sok is lost, Mok and Hok are translated and
the cell dies.
The difficulty with the hok-sok system is that a significant number
of plasmidless cells can arise even when the hok-sok system is
operative.
1.2.7.3 Balanced Lethal Systems
In a balanced-lethal system (a PSK function), a chromosomal gene
encoding an essential structural protein or enzyme is deleted from
the bacterial chromosome or is mutated such that the gene can no
longer operate. The removed or damaged gene is then replaced by a
plasmid comprising a fully operating gene and a nucleotide sequence
encoding the protein or peptide of interest, e.g., an antigen. Loss
of the plasmid results in an insufficiency of the essential protein
and the death of the plasmidless cell.
A balanced-lethal system has been successfully employed in S.
typhimurium based on expression of the asd gene encoding aspartate
.beta.-semialdehyde dehydrogenase (Asd). Asd is a critical enzyme
involved in the synthesis of L-aspartic-.beta.-semialdehyde, which
is a precursor essential for the synthesis of the amino acids
L-threonine (and L-isoleucine), L-methionine, and L-lysine, as well
as diaminopimelic acid, a key structural component essential to the
formation of the cell wall in Gram-negative bacteria. Loss of
plasmids encoding such a critical enzyme would be lethal for any
bacterium incapable of synthesizing Asd from the chromosome, and
would result in lysis of the bacterium due to an inability to
correctly assemble the peptidoglycan layer of its cell wall.
The asd system (a PSK function) has been successfully employed in
attenuated S. typhimurium-based live vector strains for
immunization of mice with a variety of procaryotic and eucaryotic
antigens including such diverse antigens as detoxified tetanus
toxin fragment C and the LT enterotoxin, synthetic hepatitis B
viral peptides, and gamete-specific antigens such as the human
sperm antigen SP10.
Murine mucosal immunization with these live vector strains has
elicited significant immune responses involving serum IgG and
secretory IgA responses at mucosal surfaces.
The asd system has recently been introduced into attenuated
Salmonella typhi vaccine strains in an attempt to increase the
stability of plasmids expressing synthetic hepatitis B viral
peptides. However, when volunteers were immunized with these live
vector strains, no immune response to the foreign antigen was
detected. In fact, to date, few reports have documented an immune
response to plasmid-based expression of a foreign antigen from
stabilized plasmids after human vaccination with an attenuated S.
typhi live vector.
In one report, the vaccine strain Ty21a was made auxotrophic for
thymine by selecting in the presence of trimethoprim for an
undefined mutation in the thyA gene, encoding thymidylate
synthetase. Although in some cases failure of live vector strains
may have resulted from over-attenuation of the strain itself, it
appears probable that current killing systems for plasmids suffer
from additional limitations.
Those situations where the chromosomal copy of the gene has been
inactivated, rather than removed, may allow for restoration of the
chromosomal copy via homologous recombination with the
plasmid-borne gene copy if the bacterial strain utilized is
recombination-proficient.
Balanced-lethal systems based on catalytic enzyme production are
subject to a number of important deficiencies. In particular, since
complementation of the chromosomal gene deletion requires only a
single gene copy, it is inherently difficult to maintain more than
a few copies of an expression plasmid. The plasmidless host strain
must be grown on special media to chemically complement the
existing metabolic deficiency.
Plasmidless cells may also benefit from "cross-feeding" effects
when a diffusible growth factor is growth limiting.
There is therefore a need in the art for a Plasmid Maintenance
System which is not solely reliant on a balanced lethal system,
particularly for use in bacterial live vector vaccines.
2. SUMMARY OF THE INVENTION
The present invention relates generally to a stabilized expression
plasmid which carries a Plasmid Maintenance System and a nucleotide
sequence encoding a protein or peptide, such as a foreign antigen,
and methods for making and using the stabilized expression
plasmids.
In a particular aspect, the stabilized expression plasmid is
employed in a Salmonella typhi live vector vaccine, such as the
strain CVD908-htrA.
The invention optimizes the maintenance of expression plasmids at
two independent levels by: (1) removing sole dependence on balanced
lethal maintenance systems; and (2) incorporating a plasmid
partition system to prevent random segregation of expression vector
plasmids, thereby enhancing their inheritance and stability. In one
aspect of the invention, the stabilized expression plasmid is
recombinantly engineered to express one or more antigens,
preferably one or more Shiga toxin 2 (Stx2) antigens, such as Shiga
toxin subunit pentamers or a genetically detoxified Stx 2.
The stabilized expression plasmid preferably comprises one or more
non-catalytic Plasmid Maintenance Functions.
In another aspect, the expression plasmid comprises a Plasmid
Maintenance System which comprises at least one PSK function and at
least one SEG function. For example, the Plasmid Maintenance System
may comprise a two-component Plasmid Maintenance System comprising
one PSK function and one SEG function. Alternatively, the Plasmid
Maintenance System may comprise a three-component Plasmid
Maintenance System comprising a PSK function, a SEG function and
another Plasmid Maintenance Function. In a preferred alternative,
the Plasmid Maintenance System comprises hok-sok+par+parA.
The Plasmid Maintenance Systems can be incorporated into multicopy
expression plasmids encoding one or more proteins or peptides of
interest. Such multicopy expression plasmids produce a gene dosage
effect which enhances the level of expression of the protein or
peptide of interest. Where the Plasmid Maintenance System is to be
employed in a bacterial live vector vaccine, the protein or peptide
of interest is one or more foreign antigens.
In one aspect, the expression plasmid is a vaccine expression
plasmid comprising a Plasmid Maintenance System and at least one
antigen, for example, at least one Shiga toxin 2 (Stx2) antigen.
Where the antigen is a Shiga toxin 2 antigen, the Shiga toxin 2
antigen can, for example, be either a B subunit pentamer or a
genetically detoxified Stx 2.
In another aspect the expression plasmid comprises a Plasmid
Maintenance System which incorporates the ssb balanced lethal
system and the ssb locus of the bacterial live vector has been
inactivated using a suicide vector comprising a temperature
sensitive origin of replication. In one aspect, the bacterial live
vector is S. typhi and the suicide vectors are used to inactivate
the ssb locus of S. typhi. In one aspect, the suicide vector is a
derivative of pSC101 which carries sacB, described herein.
In another aspect, the present invention provides a Plasmid
Maintenance System incorporating a PSK function involving a silent
plasmid addiction system based on antisense RNA control mechanisms
that only synthesize lethal proteins after plasmid loss has
occurred.
In one aspect the expression plasmid comprises a series of
expression plasmids, each comprising self-contained genetic
cassettes encoding regulated expression of a heterologous antigen,
an origin of replication, and a selectable marker for recovering
the plasmid.
In one aspect the expression plasmid comprises a Plasmid
Maintenance System which incorporates a PSK function based on the
ssb gene. In a related aspect, mutated alleles such as ssb-1,
described herein, are incorporated into the expression plasmids to
enhance higher copy number plasmids by overexpression of SSB1-like
proteins to form the required biologically active tetramers of
SSB.
In another aspect, the expression plasmid comprises a promoter. The
promoter is preferably an inducible promoter, such as the ompC
promoter. In one aspect, the inducible promoter is the mutated
P.sub.ompC1 promoter described herein.
In one aspect, the expression plasmid of the present invention
comprises a plasmid inheritance (or partition) locus; an origin of
replication selected to provide copy number which effectively
stabilizes a given antigen; a PSK function; and a nucleotide
sequence encoding an antigen and a promoter which controls
translation of the antigen and has a strength which is selected to
improve antigen production without killing the cell.
The present invention also provides a method of using the
expression plasmid comprising transforming a bacterial cell using
said expression plasmid, and culturing the bacterial cell to
produce the protein or peptide (e.g., the antigen), and/or
administering said transformed cell or cell culture to a subject.
Where the transformed bacterial cells are administered to a
subject, they are administered in an amount necessary to elicit an
immune response which confers immunity to the subject for the
protein or peptide. The subject is preferably a human, but may also
be another animal, such as a dog, horse, or chicken.
In one aspect, an expression plasmid is provided which comprises at
least 3 independently functioning expression cassettes wherein one
cassette encodes a protein or peptide of interest and the remaining
cassettes each encode a different Plasmid Maintenance Function.
In one aspect, an expression plasmid is provided which encodes (1)
a test antigen operably linked to a promoter and (2) a Plasmid
Maintenance System.
In another aspect, a regulated test antigen expression cassette is
provided which operates such that as induction of antigen
expression is increased, a metabolic burden is placed on the
bacterium which leads phenotypically to plasmid instability, i.e. a
selective advantage is created for all bacteria which can
spontaneously lose the offending plasmid. The test antigen can be
the green fluorescent protein (GFP). The expression cassette
encoding the test antigen can also comprise an inducible promoter,
such as the ompC promoter, positioned such that the inducible
promoter drives the translation of the test antigen.
In one aspect, a method of making an expression plasmid is provided
which comprises synthesizing an expression plasmid comprising at
least 3 independently functioning expression cassettes wherein one
cassette encodes a protein or peptide of interest and the remaining
cassettes each encode a different Plasmid Maintenance Function.
In one aspect, a method of screening Plasmid Maintenance Systems is
provided comprising: providing one expression cassette which
encodes a protein or peptide of interest, and at least two other
expression cassettes, each encoding and capable of expressing in
the host bacterial live vector a different Plasmid Maintenance
Function; inserting the three expression cassettes into a single
expression plasmid; transforming a bacterial live vector with the
single expression plasmid; culturing the transformed bacterial live
vector; and determining the rate of introduction of plasmidless
cells into the culture.
In one aspect, the present invention comprises an attenuated
bacterial live vector vaccine comprising an attenuated bacterial
live vector which has been transformed with a stabilized expression
plasmid comprising a Plasmid Maintenance System, preferably a
non-catalytic plasmid maintenance system.
In one aspect, the present invention comprises an attenuated
bacterial live vector vaccine comprising an attenuated bacterial
live vector which has been transformed with an expression plasmid
comprising a Plasmid Maintenance System which incorporates at least
one PSK system and one SEG system. The attenuated bacterial live
vector can, for example, be S. typhi CVD908-htrA.
The present invention also provides a method for vaccinating a
subject comprising administering to the subject an amount of a
bacterial live vector vaccine sufficient to elicit an
immunity-enhancing immune response. The present invention also
provides a method for preventing a disease by vaccinating a subject
using an amount of such bacterial live vector sufficient to elicit
an immune response to one or more pathogens of such disease. The
subject is preferably a human but may also be another animal, such
as a horse, cow or pig. For example, the present invention provides
a method for preventing hemolytic uremic syndrome (HUS) caused by
Shiga toxin 2-producing enterohemorrhagic Escherichia coli by
administering to a subject an amount of a bacterial live vector
transformed with a stabilized plasmid encoding at least one Shiga
toxin 2 antigen.
In another aspect, the present invention provides a method for
screening Plasmid Maintenance Systems for efficacy, the method
comprising: providing expression plasmids comprising the Plasmid
Maintenance Systems described herein and encoding for a protein or
peptide of interest, said expression plasmids having copy numbers
which vary from low copy number (i.e. .about.5 copies per cell) to
medium copy number (.about.15 copies per cell) to high copy number
(.about.60 copies per cell); transforming bacterial live vectors
with such expression plasmids; and testing for rate of introduction
of plasmidless cells and/or rate of growth of plasmid-containing
cells. The modified origins of replication may be origins of
replication from the plasmids pSC101 (low copy number), pACYC184
(medium copy number), and pAT153 (high copy number). Independently
functioning plasmid replication cassettes can be utilized which
permit testing of the efficiency of one or more plasmid
stabilization systems as copy number is increased.
In another aspect, the present invention provides stabilized
expression plasmids for use in attenuated S. typhi live vectors
which contain a selectable marker which can readily be replaced by
a non-drug resistant locus or by a gene encoding an acceptable drug
resistance marker such as aph encoding resistance to the
aminoglycosides kanamycin and neomycin.
The constructs of the present invention provide improved stability
of recombinant plasmids, overcoming prior art problems of plasmid
instability, for example, in bioreactor and live vector vaccination
uses. The plasmids of the present invention are specifically
tailored for vaccine applications though such plasmids are also
useful in large scale protein production.
The plasmids of the present invention are a major improvement over
the prior art in that they overcome the problems associated with
plasmidless takeover and plasmid instability and have wide ranging
utility in fields such as commercial protein production and
attenuated bacterial live vector vaccine production.
There has long been a need for a solution to the problems of
plasmidless takeover and plasmid stability associated with the
field of vaccine delivery and protein production. The present
invention is a major step toward the satisfaction of this need.
3. DEFINITIONS
The term "Plasmid Maintenance System" ("PMS") as used herein refers
to a nucleotide sequence comprising at least one post-segregational
killing function ("PSK") and at least one partitioning or
segregating system ("SEG").
The term "Plasmid Maintenance Function" is used herein to refer to
any function associated with a PMS.
The term "Post-Segregational Killing System" (PSK) is used herein
to refer to any function which results in the death of any newly
divided cell which does not inherit the plasmid of interest, and
specifically includes balanced-lethal systems such as asd or ssb,
proteic systems such as phd/doc and antisense systems such as
hok-sok.
The terms "Segregating System" and/or "Partitioning System" (both
referred to herein as "SEG") are used interchangeably herein to
refer to any Plasmid Maintenance function that operates to increase
the frequency of successful delivery of a plasmid to each newly
divided cell, as compared to the frequency of delivery of a
corresponding plasmid without such a SEG system. SEG systems
include, for example, equipartitioning systems, pair-site
partitioning systems, and the par locus of SC101.
The term "detoxified" is used herein to describe a toxin having one
or more point mutations which render the toxin non-toxic versus a
corresponding toxin without such point mutations.
The term "immunizingly effective" is used herein to refer to an
immune response which confers upon the subject of such immune
response a degree of immunological cellular memory with the effect
that a secondary response (to the same or a similar toxin) is
characterized by one or more of the following characteristics:
shorter lag phase in comparison to the lag phase resulting from a
corresponding exposure in the absence of immunization, production
of antibody which continues for a longer period than production of
antibody for a corresponding exposure in the absence of such
immunization, a change in the type and quality of antibody produced
in comparison to the type and quality of antibody produced from
such an exposure in the absence of immunization, a shift in class
response, with IgG antibodies appearing in higher concentrations
and with greater persistence than IgM, an increased average
affinity (binding constant) of the antibodies for the antigen in
comparison with the average affinity of antibodies for the antigen
from such an exposure in the absence of immunization, and/or other
characteristics known in the art to characterize a secondary immune
response.
4. BRIEF DESCRIPTION OF THE DRAWINGS
FIGS. 1A, 1B and 1C: Genetic maps of exemplary pGEN expression
plasmids (pGEN2, pGEN3, and pGEN4) of the present invention.
FIGS. 2A, 2B, 2C and 2D: Genetic maps of exemplary expression
plasmids (pJN72, pJN51, pJN10, and pJN12) of the present
invention.
FIGS. 3A-H: Flow cytometry histograms of GFP flourescence for CVD
908htrA carrying expression vectors with the hok-sok
post-segregational killing system.
FIGS. 4a-d: pGEN2 (SEQ. ID. NO.1) nucleotide sequence 1-4199.
FIGS. 5a-b: pGEN3 (SEQ. ID. NO.2) nucleotide sequence 1201-2400
showing the sequence of ori15A.
FIGS. 6a-e: pGEN4 (SEQ. ID. NO.3) nucleotide sequence 1201-3850
showing the sequence of ori101.
5. DETAILED DESCRIPTION OF THE INVENTION
Bacterial live vector vaccines employs a bacterial live vector to
express genes encoding protective antigens of bacterial, viral or
parasitic pathogens. The bacterial protective antigens are
preferably non-native to the bacterial live vector, i.e.
heterologous. The bacterial live vector vaccine is administered to
a host, thereby exposing the expressed antigens to the host's
immune system, eliciting an immune response of appropriate
character to confer immunity on the host.
In order to achieve enhanced immunogenicity, the plasmids
expressing such protective antigens must be stabilized. To the
inventor's knowledge, no currently available S. typhi-based Plasmid
Maintenance System takes advantage of naturally occurring partition
mechanisms known to improve the stability of multicopy
plasmids.
The present invention provides a non-catalytic Plasmid Maintenance
System for the stabilization of expression plasmids encoding
foreign antigens in a S. typhi live vector vaccine strain. In one
aspect the S. typhi strain is CVD 908-htrA. In another aspect, the
present invention improves and/or optimizes maintenance of
expression plasmids by providing Plasmid Maintenance Systems which
operate at two independent levels: (1) removing sole dependence on
catalytic balanced lethal maintenance systems; and (2)
incorporating a plasmid partition system which will remove random
segregation of the expression plasmids, thereby enhancing their
inheritance and stability.
The non-catalytic Plasmid Maintenance System of the present
invention improves the stability of multicopy expression plasmids
within a live bacterial vector vaccine, such as CVD908-htrA.
In one aspect, the present invention incorporates the naturally
occurring PSK function hok-sok from the antibiotic-resistance
factor pR1 within multicopy expression plasmids. The hok-sok system
is a silent plasmid addiction system based on antisense RNA control
mechanisms that only results in synthesis of lethal proteins after
plasmid loss has occurred. A critical reason for pursuing this
particular approach is that this method of improving plasmid
maintenance involves no additional manipulations of the live vector
strain, and therefore can improve the immunogenicity of
heterologous antigens expressed within any live vector strain.
The present invention also provides a plasmid maintenance system
comprising a complementation-based PSK function in which the
chromosomal gene ssb, encoding the essential non-catalytic
single-stranded binding protein (SSB) required for DNA replication,
is specifically deleted and inserted within a multicopy expression
plasmid.
The present invention also provides an improved Plasmid Maintenance
System comprising an expression plasmid having inserted therein a
SEG locus and PSK function.
5.1 Suicide Vectors
Heterologous antigens can be expressed within live vector strains,
such as CVD908-htrA, from genes residing either on plasmids or
integrated within the chromosome. One technique for integrating
these genes into the host chromosome involves the use of
temperature sensitive "suicide vectors" such as pIB307 which
contains a temperature-sensitive origin of replication from pSC101
(ori101). The present invention provides an improved suicide vector
for use in CVD908 and CVD908-htrA, derived from pIB307 which allows
for easier construction of mutagenesis cassettes to alter the live
vector chromosome.
Integration of these suicide vectors into the chromosome by
homologous recombination results from temperature inactivation of
the plasmid replication protein, RepA, a protein essential to the
function of ori101. Spontaneous resolution of the resulting
unstable merodiploid intermediates is detected by counter-selection
for loss of the sacB gene contained on the resolving suicide
vector. The sacB gene contained on all excised plasmids encodes the
levansucrase enzyme, which is lethal when expressed within the
cytoplasm of enteric bacteria, including S. typhi, growing in the
presence of sucrose. Since resolving merodiploids are selected by
incubating in the presence of 10% sucrose, excised plasmids will
kill host bacteria unless they cure spontaneously.
This system was successfully used to integrate a
kanamycin-resistance cassette into the .DELTA.aroC1019 locus of
CVD908. However, the success of these experiments rested on the
fact that the gene being mobilized into the chromosome of S. typhi
encoded a selectable drug-resistance marker. Using these early
vectors, replacing the kanamycin-resistance cassette with a
non-selectable marker was not successful because, although the
incoming marker could be integrated into the chromosome as a
merodiploid, resolution of the merodiploid to replace the drug
resistance gene was never detected.
The present invention also provides a method for using such suicide
vectors to inactivate the ssb locus of attenuated Salmonella typhi
strains such as CVD908.-htrA.
The present invention allows such suicide vectors to permit
efficient mobilization of genes expressing proteins or peptides of
interest, such as heterologous antigens, into the chromosome of S.
typhi CVD908-htrA in two stages. A sacB-aph cassette is first
introduced into the .DELTA.aroC1019 locus which was selected for
using kanamycin. Generation of this S. typhi
CVD908-htrA.DELTA.aroC1019::sacB-aph strain creates a valuable
intermediate strain into which, in theory, any structural gene can
be efficiently inserted into the aroC locus by marker-exchange. The
sacB gene is used as a counter-selectable marker by passing
merodiploids in the presence of 10% sucrose to select for
replacement of the sacB-aph cassette with the incoming antigen
cassette, since resolution of merodiploids in the presence of
sucrose will result in loss of the sacB gene in order to produce
viable progeny. This intermediate strain was employed to
efficiently integrate the non-toxigenic mutant LT-K63 of the E.
coli heat-labile enterotoxin, creating
CVD908.DELTA.aroC1019::LT-K63.
5.2 Plasmid-Based Expression of Heterologous Antigens
Although chromosomal integration of foreign genes confers stability
to such sequences, the genetic manipulations involved can be
difficult, and the drop in copy number of the heterologous gene
often results in production of insufficient levels of heterologous
antigen to ensure an optimal immune response.
In contrast, plasmid stability is a complex phenomenon which
depends on multiple factors including (1) copy number of the
plasmid; (2) appropriately regulated expression of genes contained
within the plasmid; and (3) selective pressure for ensuring the
proper segregation and inheritance of the plasmid.
To ensure stability, plasmids must be replicated in a regulated
manner to prevent their copy number from rising to lethal
levels.
In addition, plasmids must segregate during the division of a
growing bacterium to ensure that each daughter cell receives at
least one copy of the plasmid. Segregation can be a passive, random
event or an active process involving synthesis of novel proteins
which aid in plasmid segregation and inheritance. Successful
inheritance of randomly segregating plasmids relies on a high
enough copy number of randomly distributed plasmids within a
dividing bacterium to virtually guarantee inheritance of at least
one plasmid by each daughter cell.
The commonly used plasmid cloning vectors, including medium copy
number pBR322 derivatives and high copy number pUC plasmids, are
inherited by random segregation.
Active segregation involves the synthesis of proteins which are
proposed to bind to such plasmids and further coordinate with the
membranes of dividing bacteria to ensure that each daughter
receives at least one plasmid copy. Plasmids employing such active
partitioning systems are typically very low copy number plasmids
such as the F sex factor of E. coli or antibiotic resistance
R-factors such as pR1 and pRK2.
The present invention exploits naturally occurring SEG functions to
enhance inheritance of multicopy expression plasmids, which would
otherwise be inherited by random segregation, to increase the
stability of these plasmids.
The present invention also takes advantage of other naturally
occurring genetic systems in which daughter cells which do not
successfully inherit an expression plasmid will be killed and
removed from the growing population, i.e., PSK functions. The
incorporation of more than one category of plasmid stabilization
function is referred to herein as a Plasmid Maintenance System. For
example, the incorporation of both a SEG function such as a
partition locus and a PSK function into a single expression plasmid
yields a Plasmid Maintenance System.
It should be noted that a gene conferring resistance to a
bactericidal antibiotic, such as the aph gene encoding resistance
to kanamycin and neomycin, is also considered a PSK function, as is
the asd-based balanced-lethal system.
5.3 Balanced Lethal Systems
One method of ensuring the inheritance of expression plasmids
involves the construction of a PSK function referred to as a
balanced lethal system for plasmids expressing heterologous
antigens. In a plasmid-based balanced lethal system, plasmids
replicating in the cytoplasm of the bacterium express a critical
protein required by the bacterium to grow and replicate. Loss of
such plasmids removes the ability of the bacterium to express the
critical protein and results in cell death.
Such a system has been successfully employed in S. typhimurium and
is based on expression of the asd gene encoding aspartate
.beta.-semialdehyde dehydrogenase (Asd). Asd is a critical enzyme
involved in the synthesis of L-aspartic-.beta.-semialdehyde, which
is a precursor essential for the synthesis of the amino acids
L-threonine (and L-isoleucine), L-methionine, and L-lysine, as well
as diaminopimelic acid, a key structural component essential to the
formation of the cell wall in Gram-negative bacteria. Loss of
plasmids encoding such a critical enzyme is lethal for any
bacterium incapable of synthesizing Asd from the chromosome,
resulting in lysis of the bacterium due to an inability to
correctly assemble the peptidoglycan layer of its cell wall.
The asd system for improving the stability of expression plasmids
by removing plasmid-cured bacteria from the population (i.e. a PSK
function), has been successfully employed in attenuated S.
typhimurium-based live vector strains for immunization of mice with
a variety of procaryotic and eucaryotic antigens including such
diverse antigens as detoxified tetanus toxin fragment C and the LT
enterotoxin, synthetic hepatitis B viral peptides, and
gamete-specific antigens such as the human sperm antigen SP10.
Murine mucosal immunization with these live vector strains has
elicited significant immune responses involving serum IgG and
secretory IgA responses at mucosal surfaces. The asd system has
recently been introduced into attenuated S. typhi vaccine strains
in an attempt to increase the stability of plasmids expressing
synthetic hepatitis B viral peptides.
However, when volunteers were immunized with these live vector
strains, no immune response to the foreign antigen was detected.
See Tacket et al., Infection and Immunity, 65:3381, 1997
(incorporated herein by reference). In fact, to date, few reports
have documented an immune response to plasmid-based expression of a
foreign antigen from plasmids (stabilized or otherwise) after
vaccination of humans with an attenuated S. typhi live vector.
Although in some cases failure of live vector strains may have
resulted from over-attenuation of the strain itself, the inventor's
conclusion is that currently used PSK functions for plasmids suffer
from additional limitations, in particular, from segregation
imitations and catalytic activity limitations. The present
invention provides improved expression plasmids comprising enhanced
segregation capabilities by incorporating a partitioning system
along with a PSK system.
5.4 Segregation Limitations
One limitation of plasmid maintenance functions such as the asd
function (as well as the thyA function) is that they do not enhance
the inheritance of resident plasmids, which continue to segregate
randomly with or without the presence of the asd function.
Therefore, if resident expression plasmids carrying asd genes are
inherently unstable, they will be lost, regardless of the
requirement of the bacterium for Asd.
The inherent stability of an asd expression plasmid can be defined
by growing plasmid-bearing strains in the presence of DAP, which
removes the selective pressure that ensures that all viable
bacteria contain the expression plasmid. If a given plasmid is
inherently unstable, it will be lost from bacteria at a high rate
and such plasmidless bacteria will lyse in the absence of growth
supplements; the overall result of this effect will be a population
of bacteria that grows much slower than wildtype unaltered
strains.
The present invention can improve plasmid stability by
incorporating a SEG function, such as a partition locus, onto the
expression plasmid to enhance the inheritance of such plasmids by
actively dividing bacteria. As pointed out above, partition loci
are naturally present on the virulence plasmids of S. typhimurium.
Tinge and Curtiss, Journal of Bacteriology, 172:5266, 1990
(incorporated herein by reference) reported that such partition
loci were well conserved among S. typhimurium virulence plasmids,
and that when a 3.9 kb restriction fragment encoding this locus was
introduced onto the lower copy number plasmid pACYC184 (.about.15
copies per cell), the observed plasmid stability increased from 34%
plasmid-containing cells to 99% plasmid-bearing cells after 50
generations. The nucleotide sequence of this locus was later
determined by Cerin and Hackett, Plasmid, 30:30, 1993 (incorporated
herein by reference), (GenBank Accession Number M97752).
5.5 Catalytic Activity Limitations
Another potential limitation of a plasmid maintenance function such
as the asd function (as well as the thyA system) is its reliance on
an enzyme with catalytic activity. Given that complementation with
only a single copy of the asd gene is sufficient to remove
auxotrophy, it is not clear why all copies of a multicopy plasmid
should remain stable, especially if they encode an especially
problematic heterologous antigen which inhibits growth of the
bacterium.
Further, although higher copy number expression plasmids may
express appreciable levels of a given heterologous antigen in
vitro, such plasmids may not be maintained at the expected copy
numbers in vivo due to toxicity and may in fact be present at much
lower copy numbers, which would be expected to reduce any observed
immune response specific for the heterologous antigen. Accordingly,
the present invention thus provides stably maintained low and
medium copy number plasmids for expressing heterologous
antigens.
5.6 The Non-Catalytic ssb PSK Function
The potential limitation of catalytic activity associated with
balanced lethal systems is addressed here through the use of
plasmids expressing the single-stranded binding protein (SSB) from
S. typhi to trans-complement an otherwise lethal mutation
introduced into the chromosomal ssb gene. The biochemistry and
metabolic roles of the E. coli SSB protein have been extensively
reviewed in Lohman et al., Annual Reviews in Biochemistry 63:527,
1994 and Chase et al., Annual Reviews in Biochemistry 55:103, 1986
(the disclosures of which are incorporated herein by
reference).
SSB is a non-catalytic 177 amino acid protein, with a relative
molecular weight of 19 kDa, that binds with high affinity to
single-stranded DNA (ssDNA), and plays an essential role as an
accessory protein in DNA replication, recombination, and repair.
The biologically relevant form of SSB involved in binding to ssDNA
is a tetramer, which binds in two modes to ssDNA, intimately
associating with an average of either 35 (SSB.sub.35 -binding mode)
or 65 bases (SSB.sub.65 -binding mode). The specific conditions
controlling the preferred mode of binding are complex and depend on
the surrounding concentration of monovalent and divalent salts, pH,
and temperature, as well as the amount of SSB protein present.
Under given conditions, high concentrations of SSB favor the
SSB.sub.35 -binding mode, with lower SSB concentrations favoring
the SSB.sub.65 -mode. However, it must be emphasized that in both
binding modes, the required conformation of SSB is a tetramer.
Spontaneously occurring temperature-sensitive point mutations
within the ssb gene have now been characterized at the biochemical,
physiological, and nucleotide level; one such mutant, ssb-1,
contains the point mutation His 55 to Tyr, and has been found to be
unable to assemble correctly into tetramers at non-permissive
temperatures. These mutant strains exhibit temperature-sensitive
lethal defects in DNA replication and recombination.
The segregation frequencies of plasmids carrying ssb which
complement chromosomal ssb mutations in E. coli bacteria were
examined by Porter et al. Bio/Technology 8:47, 1990 (incorporated
herein by reference). They observed that in experiments involving
bioreactors, the segregation frequency in plasmid-bearing strains
growing in continuous culture under non-selective conditions for
150 hours was less than 1.times.10.sup.-7 ; this segregation
frequency was independent of copy number, as both lower copy number
pACYC184 plasmids and very high copy number pUC19 plasmids were
maintained at the same frequency. However, it must be noted that
the plasmids involved expressed only a drug-resistance marker in
addition to the SSB protein.
The present invention provides an improved plasmid maintenance
system which incorporates a partition locus such as that present on
pSC101, and may also incorporate an active partitioning system such
as that described above for the virulence plasmid of S.
typhimurium.
The present invention removes dependence on catalytic enzymes to
confer plasmid stability. In one aspect, mutated alleles similar to
ssb-1 are introduced into the expression plasmids to enhance higher
copy number plasmids by overexpression of SSB1-like proteins to
form the required biologically active tetramers of SSB. In another
aspect the present invention provides a PSK function involving a
silent plasmid addiction system based on antisense RNA control
mechanisms that only synthesize lethal proteins after plasmid loss
has occurred.
5.7 Expression Plasmids and Self-contained Genetic Cassettes
The present invention also comprises a series of expression
plasmids which are referred to herein as pGEN plasmids. pGEN
plasmids comprise self-contained genetic cassettes encoding
regulated expression of a heterologous antigen, an origin of
replication, and a selectable marker for recovering the plasmid.
This vector series has been specifically designed to test whether
any Plasmid Maintenance System can increase the stability of
plasmids, for example within an attenuated S. typhi vaccine
background.
The basic structure of these vectors is represented in FIG. 1, and
the composite gene sequences for the vectors pGEN 2 (SEQ. ID.
NO.1), pGEN 3 (SEQ. ID. NO.2) and pGEN 4 (SEQ. ID. NO.3) are
represented in FIGS. 4, 5 and 6, respectively.
It is critical to note that the pGEN plasmids are designed to be
comprised of a set of 3 independently functioning genetic
cassettes. These cassettes have been constructed such that
individual components can be optimized by replacement as necessary.
Accordingly, in addition to the various Plasmid Maintenance Systems
described herein, the cassettes can test other promising systems
now in existence or which may become available in the future.
Further, the optimized plasmid(s) can be adapted to express
relevant protective heterologous antigens within attenuated vaccine
strains for immunization of humans.
The pGEN plasmids provide a regulated test antigen expression
cassette which operates such that as induction of antigen
expression is increased, a metabolic burden is placed on the
bacterium which leads phenotypically to plasmid instability, i.e. a
selective advantage is created for all bacteria which can
spontaneously lose the offending plasmid. Thus one aspect of the
present invention provides a conditionally unstable plasmid which
can be examined for stability as plasmid maintenance functions are
incorporated.
In a preferred mode, the regulated test antigen expression cassette
contained within the pGEN plasmids comprise the inducible ompC
promoter driving expression of a fluorescent protein such as the
green fluorescent protein (GFP), overexpression of which is toxic
to E. coli and S. typhi.
The present invention also comprises a series of plasmid replicons
having copy numbers which vary from low copy number (i.e. .about.5
copies per cell) to medium copy number (.about.15 copies per cell)
to high copy number (.about.60 copies per cell). To accomplish
this, origins of replication from the well-characterized plasmids
pSC101, pACYC184, and pAT153 have been modified using polymerase
chain reaction (PCR) techniques to create independently functioning
plasmid replication cassettes. These replication cassettes permit
testing of the efficiency of a plasmid stabilization system as copy
number is increased.
The present invention also comprises selectable expression plasmids
for use in attenuated S. typhi live vectors. These expression
plasmids contain a selectable marker which can ultimately be
replaced either by a non-drug resistant locus or by a gene encoding
an acceptable drug resistance marker such as aph encoding
resistance to the aminoglycosides kanamycin and neomycin.
To accomplish this, resistance cassettes encoding resistance to
carbenicillin and tetracycline have been constructed, with
transcription being efficiently terminated by an rrnB T1T2
terminator. A detailed description of the individual components
comprising the expression and replication cassettes follows.
5.8 Components of the Antigen Expression and Replication
Cassettes
5.8.1 Promoter
It will be appreciated by one of skill in the art that a wide
variety of components known in the art may be included in the
expression cassettes of the present invention, including a wide
variety of transcription signals, such as promoters and other
sequences that regulate the binding of RNA polymerase to the
promoter. The operation of promoters is well known in the art and
is described in Doi, Regulation of Gene Expression, Modem Microbial
Genetics pages 15-39 (1991) (the entire disclosure of which is
incorporated herein by reference). The ensuing description uses the
ompC promoter by way of example, and is not meant to delimit the
invention.
The promoter is preferably an environmentally regulatable promotor
controlled by a biologically relevant signal such as osmolarity. In
a preferred mode, the promoter is the ompC promoter. The ompC gene
encodes a porin protein which inserts as a trimer into the outer
membrane of a bacterial cell. Expression and control of ompC is
complex and has recently been reviewed in considerable detail in
Pratt et al., Molecular Microbiology 20:911, 1996 and Egger et al.,
Genes to Cells 2:167, 1997 (the disclosures of which are
incorporated herein by reference).
Synthesis of the OmpC protein is ultimately controlled at the level
of transcription by the osmolarity of the surrounding environment
such that increases in osmolarity are accompanied by increases in
the transcription of ompC. However, increases in osmolarity do not
directly mediate increases in the transcription of ompC. Rather,
the bacterium senses the surrounding osmolarity using a
two-component signal transduction system encoded by the ompB
operon. This operon is composed of two genes transcribed in the
order envZ-ompR. The envZ gene encodes a 450 amino acid (a.a.)
protein, containing two transmembrane regions, which inserts into
the bacterial inner membrane (perhaps as a dimer) with an
N-terminal 118 a.a. osmotic-sensing domain extending into the
periplasmic space and a C-terminal 270 a.a. catalytic domain
extending into the cytoplasm. The C-terminal catalytic domain
possesses both kinase and phosphatase activities which are
modulated by osmolarity such that as osmolarity increases, kinase
activity predominates, and as osmolarity drops, phosphatase
activity predominates.
EnvZ kinase activity phosphorylates aspartic acid residue 55 of the
239 a.a. cytoplasmic protein OmpR, creating OmpR-P. It is the
OmpR-P modified protein which binds to the ompC promoter and
activates transcription by RNA polymerase; therefore, as osmolarity
increases, increasing kinase activity of EnvZ produces higher
levels of OmpR-P, which in turn lead to greater transcription of
ompC. OmpR-P binds to a region of the ompC promoter spanning bases
-41 (relative to the transcriptional start site of +1) to -102,
with initial binding of OmpR-P to bases -78 through -102 being
followed by additional binding to bases extending to -41 as the
concentration of OmpR-P increases with osmolarity. In addition,
OmpR-P has been shown to bind to an AT-rich upstream region
extending back to base -405 which further enhances ompC
transcription.
In a preferred embodiment the ompC promoter fragment from E. coli
spans nucleotides +70 through -389. The promoter can direct
transcription within attenuated S. typhi strains of an antibiotic
resistance gene, such as the kanamycin resistance gene in an
osmotically sensitive manner. For example, our experiments have
demonstrated that when the concentration of NaCl in liquid growth
medium was increased from 0 mM to 300 mM, resistance to kanamycin
increased from 0 .mu.g/ml to >800 .mu.g/ml.
5.8.2 Origin of Replication
Due to varying degrees of toxicity associated with different
heterologous antigens (i.e. higher toxicity for antigens derived
from parasitic organisms such Plasmodium falciparum vs. virtually
no toxicity for the fragment C of tetanus toxin), the present
invention provides live vector vaccines which preferably express
such antigens from either low or medium copy plasmids. It will be
appreciated by one skilled in the art that the selection of an
origin of replication will depend on the degree of toxicity, i.e.,
the copy number should go down as toxicity to the bacterial strain
goes up. In a preferred mode, the Plasmid Maintenance System(s)
used are capable of stabilizing replicons of low or medium copy
numbers.
It is preferable for the origin of replication to confer an average
copy number which is between about 2 and about 75. In a preferred
mode the origin of replication is selected to confer an average
copy number which is between about 5 and about 50. More preferably
the range is from about 5 to about 45.
In one aspect, the origin of replication is from pSC101, conferring
a copy number of approximately 5 per genome equivalent.
The oriE1 locus specifies synthesis of a 555 base transcript called
RNA I and synthesis of a 110 base antisense RNA transcript called
RNA II. As RNA I is synthesized, the 5'-proximal region of the
transcript adopts a stem-loop structure composed of 3 domains which
can hybridize to a complementary stem-loop structure formed by RNA
II, resulting in a double stranded RNA-RNA structure forming which
causes plasmid replication to abort.
As synthesis of RNA I continues, generating the full-length 555
base transcript, a rearrangement of the secondary structure of the
transcript destroys the initial 3 domain stem-loop structure to
form an alternate stem-loop configuration which no longer
hybridizes to RNA II. Formation of this alternate structure allows
the transcript to hybridize to one DNA strand of the plasmid
itself, forming an RNA-DNA complex which is nicked by endogenous
RNAse H to trigger synthesis of the first DNA strand of the plasmid
and plasmid replication.
Plasmid replication is therefore controlled by synthesis of RNA I,
which undergoes a cascade of structural configurations leading to
initiation of replication. The necessary progression of the RNA I
folding cascade (and resulting replication initiation) is
interrupted by competition of the domains with RNA II. This
mechanism is essentially the same in plasmids containing either
oriE1 or ori15A.
The reason these two types of plasmids can coexist within the same
bacterium is due to sequence divergence within the region of
hybridization between RNA I and RNA II, such that the RNA II from
ori15A will not hybridize to RNA I from oriE1; this sequence
divergence also affects the stability of the RNA I: RNA II hybrid,
accounting for the differences in copy number between plasmids
carrying the oriE1 or ori15A origins of replication.
The structural organization of the origins of replication cassettes
for pSC101 (ori101; .about.5 copies per genome equivalent),
pACYC184 (ori15A derivative; .about.15 copies per genome
equivalent), and pAT153 (oriE1 derivative; .about.60 copies per
genome equivalent) are analogous in structure and function.
5.8.3 Expressed Protein or Peptide
When the expression cassette is used to screen Plasmid Maintenance
Systems, it preferably expresses a protein or peptide with no
metabolic activity. A preferred protein is the green flourescent
protein (GFP) of the bioluminescent jellyfish Aequorea Victoria, a
238 amino acid protein which undergoes a posttranslational
modification in which 3 internal amino acids (.sup.65
Ser-Tyr-Gly.sup.67) are involved in a cyclization and oxidation
reaction. The resulting fluorophore emits blue-green light
maximally at a wavelength of 509 nm upon irradiation with long-wave
ultraviolet light at a wavelength of 395 nm. In addition,
fluorescence activity is remarkably constant over a wide range of
pH from 5.5-12 and at temperatures up to 70.degree. C.
Since GFP has no known catalytic activity, the level of observed
fluorescence within individual bacteria expressing GFP can provide
a direct indication of transcription levels of the gfp gene carried
by each bacterium. Expression of the GFP protein has now been
quantitated in a variety of both prokaryotic and eukaryotic cells
and requires no additional cofactors or enzymes from A. victoria.
Fluorophore formation is apparently dependent either on ubiquitous
enzymes and cofactors, or is an autocatalytic event.
Individual bacteria expressing GFP can be quantitated either alone
or within macrophages, epithelial cell lines, and infected animal
tissues using flow cytometry. GFP fluorescence is absolutely
dependent on residues 2-232 of the undenatured protein. However,
fusion of unrelated biologically active protein domains to the
N-terminus of GFP has still resulted in fusion proteins with the
expected heterologous biological activity which continue to
fluoresce as well.
It has been confirmed by sequence analysis (Clontech) that the gfp
allele preferred here (i.e. gfpuv) expresses a GFP mutant
containing 3 amino acid substitutions (not involving the
fluorophore) which increase fluorescence 18-fold over that of
wildtype GFP.
In addition, 5 rarely used arginine codons have been optimized for
efficient expression of GFP in E. coli. Since comparison of
expression levels of various heterologous proteins in E. coli and
CVD908 has demonstrated equivalent or superior expression within
CVD908, it is expected that gfpuv will function efficiently in
CVD908-htrA.
A coding sequence is inserted in a correct relationship to a
promoter where the promoter and the coding sequence are so related
that the promoter drives expression of the coding sequence, so that
the encoded peptide or protein is ultimately produced. It will be
understood that the coding sequence must also be in correct
relationship with any other regulatory sequences which may be
present.
5.8.4 Heterologous Antigens
The expression plasmids of the present invention preferably express
an antigen for presentation to a host to elicit an immune response
resulting in immunization and protection from disease. While Shiga
toxins are presented herein as examples of antigens usefully
expressed by the vaccine expression plasmids disclosed herein, the
invention is broad in scope and encumpasses the expression of any
antigen which does not destroy the bacterial live vector and which
elicits an immune response when the bacterial live vector
containing said expression plasmid(s) is administered to a host,
i.e., a human or other animal.
The vaccine expression plasmids provided herein are used to
transform attenuated bacterial strains, preferably strains used for
human vaccination and most preferably used to transform
CVD908-htrA, and preferably encode either the B subunit of Stx2 or
a genetically detoxified Stx2 holotoxin.
A sub-set of STEC most often referred to as enterohemorrhagic E.
coli (EHEC) are capable of causing severe clinical syndromes
including hemorrhagic colitis, hemolytic uremic syndrome (HUS) and
thrombotic thrombocytopenic purpura (TTP) in a small proportion of
infected individuals, in addition to causing non-bloody diarrhea in
most others.
Hemorrhagic colitis is characterized by copious bloody diarrhea,
usually without fever or with only low-grade fever and a relative
paucity of fecal leukocytes demonstrable in the diarrheal stools.
These features differentiate hemorrhagic colitis from dysentery
caused by Shigella which is typically scanty stools of blood and
mucus, preceded by high fever and with large numbers of fecal
leukocytes visible by microscopy.
HUS, a potentially fatal disease that most often affects young
children but may afflict individuals of any age, is characterized
by the triad of microangiopathic hemolytic anemia, thrombocytopenia
and uremia. Currently in North America, HUS is the most frequent
cause of acute renal failure in infants and young children. In a
study by Siegler et. al. of 288 patients treated for postdiarrheal
HUS in Utah from 1970-1994, severe disease (defined as anuria
lasting longer than 7 days, oliguria lasting for longer than 14
days, or extrarenal structural damage such as stroke) occurred in
25% of cases and was associated with children less than two years
of age; about one third of these severe cases of HUS resulted in
death (5%) or severe sequelae including end-stage renal disease
(5%) or chronic brain damage (3-5%), with less severe chronic
problems involving hypertension, proteinuria, or azotemia.
TTP, which most often affects adults, is characterized by
neurologic complications such as stroke, in addition to
thrombocytopenia, hemolytic anemia and renal disease.
By far the most common EHEC serotype is O157:H7. Nevertheless,
other EHEC serotypes also cause HUS and hemorrhagic colitis,
including O26:H11, O111 :H8 and a number of others. EHEC strains
associated with HUS always elaborate one or more Shiga toxins and
carry a 60 MDa virulence plasmid. In addition, most also harbor a
chromosomal pathogenicity island (so-called LEE) having a set of
genes that encode the ability to attach and efface. It is well
accepted that Shiga toxins elaborated by EHEC play a key role in
the pathogenesis of hemorrhagic colitis and HUS.
As described in detail below, the Shiga toxin family is comprised
of two groups of toxins, Stx1 (which is essentially identical to
cytotoxin/neurotoxin/enterotoxin produced by Shigella dysenteriae
type 1, the Shiga bacillus) and Stx2 (which is immunologically
distinct from Stx1 and has several related variants). In the USA,
the overwhelming majority of EHEC associated with cases of HUS
express Stx2, either alone or in conjunction with Stx1.
The most important reservoir of EHEC infection are bovines. The
single most important mode of transmission of EHEC to humans is via
the consumption of under-cooked contaminated beef, most often
ground beef. Less commonly, a variety of other food vehicles and
other modes of transmission have been incriminated. Most notably,
EHEC are one of the handful of bacterial enteric pathogens, which,
like Shigella, can be transmitted by direct contact or by contact
with contaminated fomites.
There is great anticipation and optimism on the part of most
epidemiologists that irradiation of meat sold in the USA will
drastically curtail the transmission of EHEC to humans, since it
will curtail the single most important mode of transmission.
Nevertheless, certain risk groups exposed to other modes of
transmission of EHEC will not benefit from this intervention. For
example, the exposure of abattoir workers to EHEC, an occupational
hazard, occurs at a point in the meat processing cycle prior to
when irradiation would be utilized. For such special groups such as
these for whom risk will remain even after irradiation of meat
becomes commonplace, anti-EHEC vaccines can be useful. The present
invention provides vaccines against EHEC useful for the prevention
of infection (in the animal reservoirs or in humans) and for
preventing the severe complications of EHEC infection by
stimulating neutralizing Shiga antitoxin.
Studies with attenuated Vibrio cholerae O1 expressing Stx1 B
subunit have demonstrated the feasibility of eliciting neutralizing
Shiga antitoxin by mucosal immunization with live vectors. However,
since virtually all EHEC associated with HUS cases in the USA
express Stx2, alone or in conjunction with Stx1, it is preferable
that a vaccine for preventing the severe complications of EHEC
infection via elicitation of toxin-neutralizing antibodies should
stimulate anti-Stx2 as well as Stx1. It is within the broad scope
of the present invention to provide a stabilized plasmid system for
expressing Stx2 antigens, alone or in conjunction with Stx1, in
attenuated S. typhi live vector.
Other antigens which may be suitably delivered according to the
compositions and methods of the present invention include, for
example, those for hepatitis B, Haemophilus influenzae type b,
hepatitis A, acellular pertussis (.sub.ac P), varicella, rotavirus,
Streptococcus pneumoniae (pneumococcal), and Neisseria meningitidis
(meningococcal). See Ellis et al., Advances in Pharm., 39: 393-423,
1997 (incorporated herein by reference).
In one aspect, the antigens encoded by the expression plasmids of
the present invention are cancer vaccines.
In another aspect, the antigens encoded by these plasmids are
designed to provoke an immune response to autoantigens, B cell
receptors and/or T cell receptors which are implicated in
autoimmune or immunological diseases. For example, where
inappropriate immune responses are raised against body tissues or
environmental antigens, the vaccines of the present invention may
immunize against the autoantigens, B cell receptors and/or T cell
receptors to modulate the responses and ameliorate the diseases.
For example, such techniques can be efficacious in treating
myasthenia gravis, lupus erythematosis, rheumatoid arthritis,
multiple sclerosis, allergies and asthma.
5.8.4.1 The Shiga Toxin Family
Conradi in 1903 first reported that S. dysenteriae 1 produced a
powerful exotoxin. Because injection of this toxin led to hind limb
paralysis of rabbits it was originally called a neurotoxin.
Subsequently this toxin, Shiga toxin, was shown to be lethal for
certain cells in tissue culture (i.e., it was a cytotoxin). Vicari
et al. and then Keusch et al. demonstrated that it also functioned
as an enterotoxin.
Scientists now recognize the existence of a family of Shiga
cytotoxins which inhibit protein synthesis, leading to cell death
for susceptible cells. For many years after the revelation that
such toxins were produced by certain E. coli strains in addition to
the original Shiga toxin produced by Shigella dysenteriae type 1,
the nomenclature for this family of toxins was confusing. Since
early reports described the activity of these toxins on Vero cells
(a cell line derived from African green monkey kidney epithelial
cells), many investigators called them verotoxins. Others referred
to these toxins expressed in E.coli as Shiga-like toxins.
The protein toxins are collectively referred to herein as Shiga
toxins (Stx), and the genes encoding these toxins are designated as
stx with subscripts denoting the group and variant [i.e. stx.sub.1
for the Shiga toxin produced by E. coli that is essentially
identical to that of Shigella dysenteriae type 1 (stx), and
stx.sub.2, stx.sub.2c, stx.sub.2d, stx.sub.2e for the antigenically
distinct group of related toxins].
The structure, biochemistry and antigenicity of Shiga toxins are
well described in Melton-Celsa et al., Eschericia coli 0157:H7 and
Other Shiga Toxin-producing E. coli Strains, 1998; Takeda,
Bacterial Toxins and Virulence Factors in Disease, 1995; Gyles,
Canadian J. of Microbiology, 38:734, 1992; and O'Brien et al.,
Current Topics in Microbiology and Immunology, 180:165, 1992 (the
disclosures of which are incorporated herein by reference).
These Shiga cytotoxins are composed of a single catalytic A subunit
of approximately 32 kDa non-covalently associated with a pentameric
receptor binding domain of approximately 7.7 kDa B subunits. These
subunits are encoded by a single operon of the order stxA-stxB;
transcription of the stx and stx.sub.1 operons are iron-regulated
in both S. dysenteriae type 1 and E. coli, but no environmental
control signals have as yet been determined for any stx.sub.2
operon. None of these toxins is encoded on a plasmid; rather they
are phage-encoded (Stx1, Stx2, Stx2c, and Stx2d) or are
chromosomally encoded (Stx, Stx2e).
As mentioned above, all members of the Shiga toxin family are
cytolytic toxins which inhibit protein synthesis within susceptible
cells by blocking the binding of elongation factor 1-dependent
aminoacyl-tRNA to ribosomes. For all toxins identified from human
infections, penetration of susceptible cells by endocytosis follows
binding of the holotoxin to the necessary cell surface glycolipid
receptor globotriaosyl ceramide (Gb.sub.3), traffiking of the toxin
to the Golgi apparatus and endoplasmic reticulum, followed by
release into the cytoplasm. Shiga toxins are RNA N-glycosidases
which depurinate a single adenine from the 28S RNA of the
eucaryotic 60S ribosomal subunit, thus inactivating the 60S subunit
and eventually leading to cell death.
There are six prototypic members of the Shiga toxin family: Stx,
Stx1, Stx2, Stx2c, Stx2d, and Stx2e, which differ from one another
immunologically and in toxic activity. Significant detail has been
included here to provide background for understanding the
significance of point mutations discussed below, which are required
for the genetically detoxified holotoxins. The members of the Shiga
toxin family differ from one another in 3 fundamental ways, as
recently summarized by Melton-Celsa et al., Eschericia coli 0157:H7
and Other Shiga toxin-producing E. coli strains, 1998:
(1) Immunologically: The Shiga toxin family is composed of two
serogroups, Stx/Stx1 and Stx2; antisera raised against Stx/Stx1 do
not neutralize members of the Stx2 serogroup, as judged by the Vero
cell cytotoxicity assay.
(2) Structurally: Stx and Stx1 are essentially identical, differing
in a single amino acid at position 45 of the mature A subunit, and
the crystal structure for the Stx holotoxin has been solved. The
prototype Stx2 is only 55% homologous to residues of the mature A
subunit of Stx/Stx1 and 57% homologous to the mature B subunit,
which explains why antisera raised against Stx/Stx1 do not
neutralize members of the Stx2 group. Within the Stx2 group, Stx2e
is most distantly related, sharing 93% amino acid homology to the
mature A subunit of Stx2 and 84% homology to the mature B subunit;
Stx2c and Stx2d are very similar to Stx2, sharing 99-100% homology
in mature A subunit residues and 97% homology in mature B subunit
residues.
(3) Cytotoxicity: Stx2 is among the most lethal of the Shiga
toxins, with an LD.sub.50 for mice injected intraperitoneally of
0.5-2 ng. The LD.sub.50 for Stx1 and Stx2e is 200-400 ng, and 1-5
ng for Stx2d; however, Stx2d is unusual in that this toxin can
become activated by murine intestinal mucus to increase the
toxicity of the toxin, lowering the LD.sub.50 to 0.5 ng.
5.8.5 Site-Specific Mutagensis of Shiga Toxins
In one aspect, the invention provides a genetically detoxified
Shiga toxin. The detoxification is accomplished by site-specific
mutagenesis, introducing two defined and well-separated point
mutations altering critical residues within the catalytic site of
the A subunit. The invention also introduces two additional defined
and well-separated point mutations within the B subunit to alter
critical residues within the primary binding site (i.e. SITE I)
residing within the cleft formed by adjacent B subunits of the
holotoxin pentameric ring.
Prior attempts have been made to alter the lower affinity binding
SITE II. However, this binding site has only been identified from
molecular modeling studies, and is not extensively supported by
mutational studies which favor SITE I binding of the Gb.sub.3
receptor. Even if SITE II is an alternate low-affinity binding site
allowing entry of our mutant holotoxin into susceptible cells, the
inactivation of the catalytic domain will still prevent cell
death.
Based on amino acid sequence alignments, X-ray crystallography
studies, and molecular modeling studies, essential amino acids have
been identified comprising the active site within the catalytic A
subunit of Stx, as well as those residues comprising the binding
SITE I within the B subunit pentamer of Stx/Stx1. It is the
inventor's conclusion that the amino acids essential to the active
site are selected from the group consisting of Tyr 77, Tyr 114, Glu
167, Arg 170, and Trp 203. The residues believed to be required for
receptor binding to the clefts formed by adjacent B subunits
include Lys 13, Asp 16, Asp 17, Asp 18, Thr 21, Phe 30, Glu 28, Gly
60, and Glu. These site predictions are now being supported by
functional studies and in vivo experiments using defined single and
double mutations, within individual domains of the holotoxin,
introduced by site-specific mutagenesis. A summary of promising
mutations is presented in Table 1. Based on these data and
crystallographic predictions, it is within the broad practice of
the invention to provide expression plasmids encoding, Shiga toxins
having two specific sets of point mutations within both the A and B
subunits to create non-toxic mutant Stx2 holotoxins for use as
vaccines, such as by expression within CVD908-htrA.
TABLE 1 SITE-SPECIFIC MUTAGENESIS STUDIES DROP IN DROP IN
NEUTRALIZING SUBUNIT TOXIN MUTATION CYTOTOXICITY LETHALITY
ANTIBODIES A Stx1 Leu201 .fwdarw. Val + .DELTA. NO cytotoxicity --
-- of residues 202- 213 Stx1 Glu167 .fwdarw. Asp 10.sup.3 -- --
Stx1 Arg170 .fwdarw. Leu 10.sup.3 -- -- Stx2 Glu167 .fwdarw. Asp
10.sup.3 -- -- Stx2e Glu167 .fwdarw. Asp 10.sup.4 -- -- Stx2e
Arg170 .fwdarw. Lys 10.sup. -- -- Stx2e Glu167 .fwdarw. Asp
10.sup.4 -- -- Arg170 .fwdarw. Lys Stx2e Glu167 .fwdarw. Gln
10.sup.6 10.sup.4 Y B Stx Asp16 .fwdarw. His + NO cytotoxicity --
-- Asp17 .fwdarw. His Stx Arg33 .fwdarw. Cys 10.sup.8 -- -- Stx
Gly60 .fwdarw. Asp 10.sup.6 -- -- Stx1 Phe30 .fwdarw. Ala 10.sup.5
10.sup. Y Stx2 Ala42 .fwdarw. Thr 10.sup.3 -10.sup.4 Y Y Stx2 Gly59
.fwdarw. Asp 10.sup.3 -10.sup.4 Y Y
5.9 Pharmaceutical Formulations
It is contemplated that the bacterial live vector vaccines of the
present invention will be administered in pharmaceutical
formulations for use in vaccination of individuals, preferably
humans. Such pharmaceutical formulations may include
pharmaceutically effective carriers, and optionally, may include
other therapeutic ingredients, such as various adjuvants known in
the art.
The carrier or carriers must be pharmaceutically acceptable in the
sense that they are compatible with the therapeutic ingredients and
are not unduly deleterious to the recipient thereof. The
therapeutic ingredient or ingredients are provided in an amount and
frequency necessary to achieve the desired immunological
effect.
The mode of administration and dosage forms will affect the
therapeutic amounts of the compounds which are desirable and
efficacious for the vaccination application. The bacterial live
vector materials are delivered in an amount capable of eliciting an
immune reaction in which it is effective to increase the patient's
immune response to the expressed mutant holotoxin or to other
desired heterologous antigen(s). An immunizationally effective
amount is an amount which confers an increased ability to prevent,
delay or reduce the severity of the onset of a disease, as compared
to such abilities in the absence of such immunization. It will be
readily apparent to one of skill in the art that this amount will
vary based on factors such as the weight and health of the
recipient, the type of protein or peptide being expressed, the type
of infecting organism being combatted, and the mode of
administration of the compositions.
The modes of administration may comprise the use of any suitable
means and/or methods for delivering the bacterial live vector
vaccines to a corporeal locus of the host animal where the
bacterial live vector vaccines are immumostimulatively
effective.
Delivery modes may include, without limitation, parenteral
administration methods, such as subcutaneous (SC) injection,
intravenous (IV) injection, transdermal, intramuscular (IM),
intradermal (ID), intraperitoneal (IP), as well as non-parenteral,
e.g., oral, nasal, intravaginal, pulmonary, opthalmic and/or rectal
administration.
The dose rate and suitable dosage forms for the bacterial live
vector vaccine compositions of the present invention may be readily
determined by those of ordinary skill in the art without undue
experimentation, by use of conventional antibody titer
determination techniques and conventional
bioefficacy/biocompatibility protocols. Among other things, the
dose rate and suitable dosage forms depend on the particular
antigen employed, the desired therapeutic effect, and the desired
time span of bioactivity.
The bacterial live vector vaccines of the present invention may be
usefully administered to the host animal with any other suitable
pharmacologically or physiologically active agents, e.g., antigenic
and/or other biologically active substances.
Formulations of the present invention can be presented, for
example, as discrete units such as capsules, cachets, tablets or
lozenges, each containing a predetermined amount of the vector
delivery structure; or as a suspension.
6. EXAMPLES
An expression plasmid composed of individual cassettes has been
constructed for use in bacterial live vector vaccines such as E.
coli and Salmonella. With the exception of ribosomal binding sites
(RBS), the key genetic loci controlling transcription initiation
and termination, plasmid replication, or encoding expressed
proteins are contained within defined restriction fragments, as
depicted by the representative plasmid diagram of pGEN2 (SEQ. ID.
NO.1) seen in FIG. 1. The basic structure of these expression
plasmids will first be highlighted and then the data demonstrating
the function of each locus within the attenuated vaccine strain
CVD908-htrA will be summarized.
6.1 pGEN Structure
Transcription of any heterologous antigen to be expressed within
CVD908-htrA is primarily controlled by an inducible promoter
contained on an EcoRI-BglII cassette. Since our expression plasmids
were initially modeled after pTETnir15, early versions carried the
anaerobically-activated nir15 promoter (P.sub.nir15). However, this
promoter has been replaced with a more tightly regulated
osmotically controlled promoter P.sub.ompC which is easily
manipulated in vitro by varying the concentration of NaCl.
Heterologous antigens are contained on a BglII-AvrII cassette,
flanked by an optimized RBS at the 5'-proximal end and a trpA
transcriptional terminator at the 3'-distal end of this cassette.
The origin of replication for these expression plasmids has been
designed as an AvrII-BglII cassette, and is protected from
read-through transcription originating in flanking regions. These
cassettes carry an extremely efficient T1T2 transcriptional
terminator at one terminus with the trpA transcriptional terminator
from the heterologous antigen cassette at the opposite end of the
replication cassette.
The flanking BglII and SpeI sites between the replication cassette
and the selection cassette are intended for insertion of a plasmid
maintenance function, such as the par locus from pSC101. The
selection cassettes contained within the plasmids are contained
within SpeI-XbaI cassettes, and can, for example, be used to encode
resistance to carbenicillin (the bla gene) or resistance to
tetracycline (the tetA gene).
The drug resistance cassette can be replaced with the ssb gene
encoding the essential single stranded binding protein of
Salmonella typhi CVD908-htrA.
The flanking XbaI and EcoRI sites between the selection cassette
and P.sub.ompC are intended for insertion of a PSK locus such as
hok-sok.
6.2 P.sub.ompC.
An inducible promoter has been constructed to control expression of
a heterologous antigen for introduction to the human immune system
using the ompC promoter (P.sub.ompC)from E. coli. The basic
sequence of the ompC promoter is described in Norioka et al.,
Journal of Biologoical Chemistry, 261:17113, 1986 (the disclosure
of which is incorporated herein by reference). Synthesis was
carried out using synthetic primers designed to introduce a
5'-proximal EcoRI restriction site and 3'- distal BglII site
flanking a fragment of 465 base pairs in which the natural RBS has
been removed.
To confirm that this promoter is osmotically controlled within
CVD908-htrA, a pBR322-derived plasmid was constructed in which tetA
was replaced by a cassette comprised of P.sub.ompC driving
expression of a promoterless aph cassette derived from the sacB-neo
genes of the suicide vectors described above) which confers
resistance to kanamycin. This plasmid designated pKompC was
introduced into CVD908-htrA by electroporation, and recipients were
screened for resistance to kanamycin on LB medium. The osmotically
regulated expression of aph was determined by growing
CVD908-htrA(pKompC) in LB broth supplemented with 0.0001% (w/v)
2,3-dihydroxybenzoic acid (DHB) and 50 .mu.g/ml of kanamycin for
approximately 2 hrs to provide a seed culture; 50 .mu.l of this
culture were inoculated into 50 ml Nutrient Broth (NB) supplemented
with DHB as above, but with increasing concentrations of kanamycin;
a parallel set of cultures were set up with the identical ranges of
kanamycin added, but also containing 10% sucrose to hopefully
induce P.sub.ompC. Cultures were incubated overnight at 37.degree.
C., and the O.D..sub.600 was measured. Results are reported in the
Table 2 below for Experiment 1.
TABLE 2 Experiment 1 CONCENTRATION of CONTROL 10% SUCROSE KANAMYCIN
(.mu.g/ml) (O.D..sub.600) (O.D..sub.600) 0 0.91 0.34 50 0.13 0.35
100 0.07 0.31 300 0.02 0.19 Experiment 2 CONCEN-TRATION of CONTROL
300 mM NaCl KANAMYCIN (.mu.g/ml) (O.D..sub.600) (O.D..sub.600) 0
0.95 1.04 200 0.04 0.99 400 0.01 0.96 800 0.01 0.92
Although 10% sucrose has an inhibitory effect on the growth of
CVD908-htrA(pKompC), regardless of selective pressure using
kanamycin, it is concluded that E. coli P.sub.ompC is indeed
inducible when driving aph gene expression within
CVD908-htrA(pKompC).
To confirm this, a culture of CVD908-htrA(pKompC) in supplemented
LB broth and kanamycin was incubated for 16 hr at 37.degree. C.,
diluted 1:10 into fresh medium, and incubated at 37.degree. C. for
two hrs to provide a seed culture of exponentially growing
bacteria. 100 .mu.l aliquots of this culture were then inoculated
into 50 ml NB broth cultures (1:500 dilution) containing increasing
concentrations of kanamycin from 200 to 800 .mu.g/ml; a parallel
set of cultures were set up containing 300 mM NaCl, and all
cultures were incubated at 37.degree. C. for 16 hr. Results are
reported in Table 2 above for Experiment 2.
It is clear from these experiments that P.sub.ompC -driven
expression of the aph gene within CVD908-htrA confers resistance to
kanamycin at levels up to 800 .mu.g/ml in an osmotically regulated
manner.
6.3 Modified ompC Promoter
As described above, early versions of the expression plasmid
carried P.sub.ompC driving transcription of the aph gene. This
cassette was later replaced with a 756 bp BglII-NheI cassette
containing the gfpuv allele from pGFPuv (Clontech) and the desired
construct was recovered in E. coli. During the visual screening of
E. coli colonies sub-illuminated with ultraviolet light, one very
brightly fluorescing colony and another representative fluorescent
colony were chosen for further study, designated clone 1 and clone
2 respectively. Upon purification of the plasmids involved, it was
determined that clone 1 contained a plasmid that no longer carried
a BglII site separating P.sub.ompC from gfpuv, while clone 2
carried the expected BglII site.
The induction of GFP expression when clones 1 and 2 are grown on
nutrient agar in the presence or absence of NaCl was examined, and
it was determined by visual inspection that clone 2 displays very
little fluorescence when grown on nutrient agar containing no NaCl
but fluoresces brightly when plated on nutrient agar containing 300
mM NaCl; clone 1, however, has a higher background level of
fluorescence when uninduced and fluoresces intensely when induced
with 300mM NaCl.
To rule out mutations within the gfpuv gene which might affect
fluorescence, P.sub.ompC from clone 1 was replaced with P.sub.ompC
from clone 2, and the expected decrease in fluorescence as judged
by sub-illumination was confirmed. It was therefore concluded that
differences in observed fluorescence were controlled by two
genetically distinct versions of our P.sub.ompC promoter, which
will now be designated as P.sub.ompC (higher transcription levels
with less osmotic control) and P.sub.ompC (moderate transcription
levels with osmotic control similar to that observed for the
P.sub.ompC -aph cassette described above). The plasmids containing
these expression cassettes are designated as pGFPompC1 and
pGFPompC2, respectively.
Flow cytometry was also used to characterize differences in induced
and uninduced expression of gfpuv, controlled by P.sub.ompC and
P.sub.ompC. To accomplish this, isolated colonies of CVD908-htrA,
CVD908-htrA(pGFPompC1) and CVD908-htrA(pGFPompC2) grown on nutrient
agar containing DHB and 100 .mu.g/ml of carbenicillin were
inoculated into 40 ml broth cultures of the same medium, and grown
at 37.degree. C./250 rpm for 24 hr to generate seed cultures. Each
culture was then diluted 1:100 into either supplemented nutrient
broth, supplemented nutrient broth plus 10% sucrose, or
supplemented nutrient broth plus 300 mM NaCl, and grown at
37.degree. C./250 rpm for 48 hr; bacteria were then pelleted and
resuspended in 1 ml of phosphate-buffered saline (PBS, pH=7.4).
Suspensions were then diluted 1:100 in PBS, pH=7.4 and analyzed by
flow cytometry using a Coulter Epics Elite ESP with the argon laser
exciting bacteria at 488 nm and emissions detected at 525 nm.
Results are presented in Table 3 below.
TABLE 3 Low Osmolarity 10% Sucrose 300 mM NaCl Mean Fluor- Mean
Fluor- Mean Fluor- Strain O.D..sub.600 escence O.D..sub.600 escence
O.D..sub.600 escence CVD908-htrA 0.34 0.3 0.27 0.3 0.41 0.3
CVD908-htrA 0.36 18.8 0.25 38.0 0.38 39.4 (pGFPompC1) CVD908-htrA
0.32 14.3 0.23 40.6 0.35 37.5 (pGFPompC2)
These data clearly show that when driving expression of gfpuv
within the live vector strain CVD908-htrA, P.sub.ompC and
P.sub.ompC are inducible with increasing osmolarity, although the
basal level of transcription is still significant in both cases.
The results observed under conditions of low osmolarity further
support our observations using solid media that P.sub.ompC1 drives
higher heterologous antigen expression than P.sub.ompC.
6.4 Origins of Replication and Selection Cassettes
The success of expressing potentially toxic or otherwise
problematic heterologous antigens within CVD908-htrA depends on the
copy number of the expression plasmid. In addition, observed immune
responses to a given heterologous antigen are affected by the copy
number of the gene(s) encoding the antigen, with chromosomally
expressed antigens eliciting poorer immune responses when compared
to plasmid-based expression.
An optimized immune response will depend on multicopy plasmid-based
expression of the heterologous antigen(s) from plasmids with the
appropriate copy number.
Since the appropriate copy number for a given heterologous gene
cannot be known a priori, the present invention provides a set of
expression plasmids which contain the origins of replication oriE1
(amplified from pAT153; copy number .about.60), ori15A (amplified
from pACYC184; copy number .about.15), and ori101 (amplified from
pSC101; copy number .about.5). These self-contained replication
cassettes are all carried on BglII-BamHI fragments, and are
depicted for a set of 3 tetracycline-resistance expression plasmids
shown in FIG. 1.
Expression of the P.sub.ompC1 -controlled gfpuv expression cassette
contained on these expression plasmids was analyzed using flow
cytometry. These experiments were designed to detect whether
differences in the level of observed fluorescence could be
correlated with the expected copy number of a given, exression
plasmid. CVD908-htrA strains carrying pGEN2 (SEQ. I.D. NO.1), pGEN3
(SEQ. I.D. NO.2), and pGEN4 (SEQ. I.D. NO.3), were streaked onto
the rich medium SuperAgar supplemented with DHB and 20 .mu.g/ml
tetracycline where appropriate. SuperAgar was used because it is a
very rich medium (3.times. LB agar). Plates were incubated at
30.degree. C. to reduce the toxicity of GFP synthesis and allow
bacteria to grow luxuriously on the plates. Isolated colonies were
then inoculated into 45 ml of SuperBroth supplemented with DHB and
20 .mu.g/ml tetracycline where appropriate, and incubated at
37.degree. C. for 16 hr. Bacteria were concentrated by
centrifugation and resuspended in 1 ml of sterile PBS, pH=7.4, and
diluted 1:100 in PBS, pH=7.4 prior to FACS analysis. Bacteria were
analyzed by flow cytometry, as described above, for two independent
growth experiments, and results are displayed in Table 4 at the end
of this section.
These data support the conclusion that overexpression of GFP within
CVD908-htrA is toxic to the bacteria. As the theoretical copy
number increases for the plasmids pGEN4 (SEQ. I.D. NO.3), pGEN3
(SEQ. I.D. NO.2), and pGEN2 (SEQ. I.D. NO.1) expressing GFP under
identical growth conditions from the identical P.sub.ompC 1
promoter, the percentage of the growing population which fluoresces
declines. It is expected that the "dim" bacteria are not viable
bacteria and may no longer contain the expression plasmid, since
these cultures were grown in the presence of 20 .mu.g/ml
tetracycline. It is noted, however, that when streaked onto solid
medium and grown at 37.degree. C. for 24-36 hr, CVD908-htrA(pGEN2
(SEQ. I.D. NO.1)) grows poorly and fails to produce isolated
colonies, while CVD908-htrA(pGEN3 (SEQ. I.D. NO.2)) and
CVD908-htrA(pGEN4 (SEQ. I.D. NO.2)) grow quite well and produce
intensely fluorescing isolated colonies.
GFP is employed herein as representative of other problematic
heterologous antigens which would be of interest to include in a
bacterial live vector, such as the S. typhi-based live vector;
however, it will be appreciated that GFP can be replaced by any
non-metabolic protein or peptide antigen.
The data above show that although use of medium-copy expression
plasmids containing oriE1 replicons can be of use in expression of
some antigens, expression of antigens of higher toxicity will be
more successfully expressed from lower copy number plasmids which
employ origins of replication yielding average copy numbers between
2 and 30, such as ori15A or ori101 origins of replication.
TABLE 4 Mean Mean Percent Fluorescence Of Percent Fluorescence Dim
Dim Bacteria Fluorescing (Relative Strain Bacteria (Relative Units)
Bacteria Units) Experiment 1 CVD908-htrA 100 0.6 0 0 CV0908- 19.9
0.1 80.1 38.5 htrA(pGEN2) CVD908- 17.1 0.1 82.9 28.1 htrA(pGEN3)
CVD908- 12.1 0.1 88.0 22.4 htrA(pGEN4) EXPERIMENT 2 CVD908-htrA 100
0.3 0 0 CVD908- 37.2 0.3 62.8 10.1 htrA(pGEN2) CVD908- 4.9 0.2 95.1
8.28 htrA(pGEN3) CVD908- 9.4 0.3 90.6 4.25 htrA(pGEN43)
6.5 The hok-sok Antisense Post-segregational Killing Locus
Using the polymerase chain reaction, the hok-sok PSK genes were
amplified using the multiple antibiotic resistance R-plasmid pR1 as
the template in these reactions. All initial attempts to clone this
locus onto either high or medium copy number plasmids were
unsuccessful. In order to directly select for the hok-sok locus
during subcloning, a set of primers was designed for use in
overlapping PCR reactions such that the final product was a
fragment containing a genetic fusion of the hok-sok locus from pR1
and a promoterless tetA gene from pBR322 encoding resistance to
tetracycline. This cassette was engineered such that transcription
of the hok gene would continue into tetA; the two loci within this
cassette were separated by an XbaI restriction site for future
manipulations.
Construction of this cassette not only allowed for direct selection
of the hok-sok locus, but also allowed for confirmation that the
PSK function would operate in S. typhi CVD908-htrA. After
electroporation of plasmids carrying the cassette into CVD908-htrA,
transformants could be selected using tetracycline. Successful
recovery of isolated colonies indicates successful synthesis of the
hok-tetA mRNA, and successful synthesis of the antisense sok RNA to
prevent translation and synthesis of Hok, which would kill the
bacteria. Recovery of the hok-sok-tetA cassette then became
straightforward, and was easily incorporated into our expression
plasmids to create the selectable marker cassette of the plasmids
pGEN2 (SEQ. I.D. NO.1), pGEN3 (SEQ. I.D. NO.2), and pGEN4 (SEQ.
I.D. NO.3) depicted in FIG. 1.
Experiments were then initiated to determine the effect of the
hok-sok PSK function on the stability of expression plasmids
containing oriE1 and the resistance marker bla encoding
.beta.-lactamase which confers resistance to carbenicillin. The
hok-sok cassette was inserted into the pAT153-based expression
plasmid pTETnir15, in which the Pnir15-toxC heterologous antigen
cassette was replaced with our P.sub.ompC1 -gfpuv cassette,
creating the plasmids pJN72 (without hok-sok) and pJN51 (with
hok-sok). An additional set of plasmids was created by replacing
P.sub.ompC1 with the weaker promoter P.sub.ompC2, creating pJN10
and pJN12; the structures of these four isogenic plasmids are
represented in FIG. 2. CVD908-htrA strains carrying either pJN72,
pJN51, pJN10, or pJN12 were streaked onto the rich medium SuperAgar
supplemented with DHB and 100 .mu.g/ml carbenicillin, and plates
were incubated as above for the pGEN plasmids at 30.degree. C. to
reduce the toxicity of GFP synthesis and allow bacteria to grow
luxuriously on the plates.
Isolated colonies were then inoculated into 45 ml of Super broth
supplemented with DHB and 100 .mu.g/ml carbenicillin and grown at
37.degree. C. for 24 hours for analysis by flow cytometry of
fluorescence. A second independent experiment was carried out
exactly as the first, except isolated colonies were suspended in
500 .mu.l of Super broth and 250 .mu.l each inoculated into 45 ml
paired Super broth cultures with or without 300 mM NaCl added to
induce the P.sub.ompC -gfpuv cassette; cultures were incubated at
37.degree. C. for 48 hrs and again analyzed by flow cytometry, and
results for both experiments are displayed in Table 5. Fluorescence
histograms for uninduced and induced expression plasmids from
experiment 2 are represented in FIG. 3.
TABLE 5 Experiment 1 Mean Percent Percent Dim Fluorescence Of
Fluorescing Strain Bacteria Dim Bacteria Bacteria Mean Fluorescence
CVD908-htrA 100 0.3 CVD908- 3.1 0.2 96.9 10.2 htrA(pJN72) CVD908-
58.1 0.3 41.9 6.29 htrA(pJN51) CVD908- 5.4 0.2 94.6 7.43
htrA(pJN10) CVD908- 18.9 0.2 81.1 6.60 htrA(pJN12) Experiment 2 +/-
Mean Fluor- % 300 Mm % Dim escence Dim Fluorescing Mean Fluor-
Strain O.D..sub.600 Nacl Bacteria Bacteria Bacteria escence
CVD908-htrA 0.73 - 100 0.3 0 0 CVD908- 0.75 - 2.3 0.3 97.7 11.7
htrA(pJN72) 0.89 + 22.2 0.3 77.8 22.5 CVD908- 0.62 - 56.3 0.3 43.7
18.4 htrA(pJN51) 0.82 + 95.4 0.3 4.6 21.0 CVD908- 0.72 - 1.7 0.3
98.3 8.3 htrA(pJN10) 0.96 + 29.9 0.3 70.1 19.8 CVD908- 0.47 - 45.2
0.3 54.8 16.4 htrA(pJN12) 0.68 + 95.6 0.3 4.4 13.2
These flow cytometry results can be explained as follows:
Expression of GFPuv (or other potentially detrimental heterologous
antigen) from a multicopy expression plasmid such as pJN72
increases the metabolic stress on the CVD 908-htrA(pJN72) live
vector, and increases plasmid instability in the absence of
selection. Since the selectable marker of the expression plasmid
encodes the secreted enzyme .beta.-lactamase, then as time
increases the concentration of carbenicillin in the surrounding
medium declines, selective pressure decreases, and the frequency of
plasmid loss increases; however, since multicopy plasmids are
involved, relatively few bacteria succeed in losing all resident
plasmids, but the average copy number of pJN72 per bacterium
drops.
Quantitation by flow cytometry of GFPuv production for an uninduced
population of healthy growing CVD 908-htrA(pJN72) indicates that
the majority of bacteria express GFPuv and few non-fluorescing
cells are detected (FIG. 3, panel A). However, increasing
production of GFPuv by induction of the P.sub.ompC1 -gfpuv cassette
increases the metabolic stress on CVD 908-htrA(pJN72), and although
the production of GFP doubles, the percentage of non-fluorescent
bacteria increases as more plasmids are lost from the population
(FIG. 3B).
In a similar population of growing CVD 908-htrA(pJN51), each
bacterium carries multicopy plasmids encoding both GFPuv and a PSK
function. The frequency of plasmid loss for pJN51 remains the same
as for pJN72, but in this case as individual bacteria lose copies
of the expression plasmid, the 1:1 stoichiometry between the mRNA
levels of hok and sok is disturbed, and production of Hok leads to
cell death; therefore, the only CVD 908-htrA(pJN51) bacteria that
will grow rapidly will be those which retain all of their
expression plasmids. Accordingly, it is not surprising that
quantitation by flow cytometry of GFPuv production for an uninduced
population of healthy growing CVD 908-htrA(pJN51) now detects a
population of fluorescing bacteria which displays levels of GFPuv
fluorescence equivalent to those observed for CVD 908-htrA(pJN72)
grown under inducing conditions (FIG. 3C vs FIG. 3B); however, the
percentage of non-fluorescing bacteria rises to over half the
overall population of organisms.
Increasing production of GFPuv in this population by induction of
the P.sub.ompC1 -gfpuv cassette in CVD 908-htrA(pJN51) again
increases the metabolic stress on the live vector, but now the
percentage of non-fluorescent bacteria almost completely overtakes
the few fluorescing bacteria as many plasmids are presumably lost
from the population and bacteria are killed (FIG. 2D).
One would expect that if a weaker promoter is used to control
expression of GFPuv, the overall fluorescence of the population
would be decreased (compared to that observed for a similar
population of organisms grown with a strong promoter expressing
GFPuv under identical conditions), and the percentage of
non-fluorescent bacteria should drop due to the overall drop in
GFPuv synthesis. However, as seen in FIG. 3E-H, use of the weaker
P.sub.ompC2 -gfpuv cassette did not significantly improve the
viability of induced bacteria carrying a killing system, even
though overall expression of GFPuv was reduced.
It is concluded that in order to maximize the percentage of a
population of live vectors expressing the heterologous antigen of
choice, it is not sufficient only to incorporate a PSK function
into a given expression plasmid, whether it be a drug resistance
marker, the asd system, an alternate ssb system, or the hok-sok
killing system. In addition to optimizing copy number and
expression levels, the segregation frequencies of these plasmids
must also be improved to ensure that each daughter cell in an
actively growing population will inherit at least one expression
plasmid and those that do not will be killed and removed from the
population. It is therefore within the scope of the present
invention to provide an expression vector having a PSK function and
further having optimized copy number and/or expression levels,
coupled with incorporation of one or more SEG functions.
6.6 Complementation-based Killing System
It is also within the broad scope of the present invention to
provide an expression plasmid comprising a complementation-based
killing system, for example, a system involving the introduction of
a defined non-revertible deletion mutation into the chromosomal ssb
locus of CVD908-htrA by homologous recombination, and
trans-complementation of this lesion using multicopy plasmids
carrying ssb.
To carry out such constructions requires cloning the relevant
section of the S. typhi chromosome encompassing the ssb gene and
flanking sequences, into which specific in-frame deletions can be
introduced for chromosomal mutagensis. No complete nucleotide
sequence data have been published for the ssb gene or flanking gene
sequences within the chromosome of S. typhi; however, the
chromosomal region encompassing the ssb locus is defined within the
recently completed genomic sequence of the E. coli K12 strain
MG1655 Blattner et al., Science 277:1453, 1997 (incorporated herein
by reference).
The genomic sequence from the updated version M52 from bases U.S.
Pat. Nos. 4,268,490-4,275,593 was used as a reference sequence for
comparison against published relevant known sequences retrieved
from the GenBank database homologous to this region. This region of
the E. coli chromosome encompasses the ssb gene and establishes the
order of flanking genes as uvrA-ssb-yjcB-yjcC-soxS-soxR. Although
the functions of proteins encoded by the yjcB-yjcC loci are
unknown, the remaining genes are all essential and involved in DNA
replication, recombination, modification and repair (uvrA-ssb), or
oxidative stress (soxS-soxR).
Such essential genes as uvrA-ssb and soxS-soxR should be
significantly homologous at the nucleotide level to sequences
within the S. typhi chromosome, allowing design and synthesis of
single-stranded DNA primers for use in polymerase chain reactions
(PCR) to establish the linkage of the ssb locus within the
chromosome of CVD908-htrA. An optimal set of such primers can then
be used to amplify and clone the fragments necessary for
chromosomal mutagenesis of the S. typhi ssb gene.
Identification of potentially useful regions of homology was
attempted by alignment with the relevant chromosomal sequences from
S. typhimurium, including the uvrA-ssb locus (strain NM522; GenBank
Accession #=M93014), and soxRS (strain LT2; GenBank Accession
#=U61147). In addition, preliminary sequence data and BLAST
analysis for an S. typhi Ty2 genome sequencing project was obtained
from M. McClelland and R. Wilson at the Genome Sequencing Center,
Washington University School of Medicine in St. Louis. DNA sequence
for four clones were identified, having homology to uvrA
(hb54a04.s1), ssb (hb59e04.s1), soxS (hb53e09.s1), and a region
downstream of soxR (hb58g03.s1).
Sequence alignments and analysis using DNASIS software (Hitachi
Software) verified that the 3350 base chromosomal sequence spanning
the uvrA-ssb locus of S. typhimurium was 74.5% identical to the
same locus within E. coli; the 956 bases spanning the soxRS locus
turned out to be distantly related with only a 50.9% identity
score.
Even though the DNA sequences of the S. typhi Ty2 clones are all
short sequences of between 433 and 490 bases, regions of identity
with corresponding E. coli genes of 69% for S. typhi uvrA and 64%
for the 5'proximal 207 bases of the S. typhi ssb gene were still
identified. Although no significant homology to E. coli was
identified for the S. typhi soxR sequence, it was determined that
S. typhi soxS was .about.23% homologous to E. coli sequences and
.about.25% homologous to S. typhimurium sequences. Based on the
significant homology between the uvrA-ssb loci of E. coli, S.
typhimurium, and S. typhi, one can amplify the uvrA-ssb locus from
the chromosome of CVD908-htrA using primers homologous to well
conserved regions of the uvrA and ssb genes of E. coli.
A 3.6 kb fragment has now been successfully amplified for
purification and direct sequence analysis to define the uvrA-ssb
chromosomal locus from CVD908-htrA.
6.7 Conclusions
It is within the scope of the present invention to incorporate a
non-catalytic PSK function into expression plasmids to improve the
plasmid-based expression of a heterologous antigen within
CVD908-htrA or other attenuated live vector strain. It is also
within the scope of the invention to further incorporate at least
one partition function (preferably two partition functions) to
provide a Plasmid Maintenance System which improves and/or
optimizes the immunogenicity of S. typhi-based live vectors
delivering heterologous antigens to the human immune system. It is
also within the scope of the present invention to improve and/or
optimize the use of the hok-sok silent plasmid addiction system,
already inserted into our expression plasmids to enhance the
immunogenicity of CVD908-htrA containing expression plasmids.
Establishing this killing system involves no chromosomal
manipulation of the CVD 908-htrA live vector and can immediately be
introduced into later versions of our attenuated S. typhi vaccine
strains without further manipulation of the strain. This killing
system, coupled with at least one suitable partitioning system, can
be used as a Plasmid Maintenance System within other enteric live
vectors currently being investigated.
The hok-sok system does not introduce any other exogenous proteins
into CVD 908-htrA, to possibly affect the metabolism of the live
vector. The antisense RNA control mechanism ensures that in any
instance where the Hok protein is actually synthesized, the host
bacterium is killed and removed from the population. Expression
plasmids carrying hok-sok and either oriE1 or ori15A replicons
(which are also controlled by an antisense RNA control mechanism),
can synthesize within the live vector only the plasmid selection
protein and the heterologous antigen of choice, although it is
recognized that the expected copy number of oriE1-based expression
plasmid may be inappropriately high for some heterologous
antigens.
It is also within the scope of the present invention to provide a
new set of isogenic expression plasmids which use either the ori15A
replicon or the ori101 replicon for expression of potentially
lethal heterologous antigens. Note that expression plasmids
carrying an intact ori101 replicon will also contain the par
function which enhances plasmid segregation. The presence of the
bla gene encoding .beta.-lactamase would be undesirable in live
vector constructs for human use. In such circumstances this
cassette can be replaced with an aph cassette encoding resistance
to the aminoglycosides neomycin and kanamycin. The functional aph
cassette from the original pKompC plasmids discussed above can be
modified using PCR as an XbaI-Spe I fragment and inserted into both
pJN72 and pJN51 cleaved with XbaI-Spe I, replacing the bla cassette
in these expression plasmids, and creating pJN14 and pJN15
respectively.
It is also within the scope of the present invention to select
clones with the desired counterclockwise orientation of aph (i.e.
transcribing away from the P.sub.ompC1 -gfpuv cassette) by
screening plasmids for the presence of the unique Spe I restriction
site. pJN14 and pJN15 will then be cleaved with Avr II-Bgl II, and
the oriE1 replicons replaced with the ori15A Avr II-Bam HI cassette
from pGEN3 (SEQ. I.D. NO.2), or the ori101 Avr II-Bam HI cassette
from pGEN4 (SEQ. I.D. NO.3), creating pJN16 and pJN17. The
influence of the hok-sok locus and copy number within the isogenic
set of expression plasmids pJN14, pJN15, pJN16, and pJN17, can then
be examined as transcription levels are kept relatively constant
within the P.sub.ompC1 -gfpuv cassette. Note that since the origins
of replication within these expression plasmids are sequestered by
transcriptional termination signals at both the 5'-proximal and
3'-distal termini, variations in copy number due to read-through
transcription from other promoters within these plasmids can be
minimized. To examine the effect of promoter strength, an
additional set of analogous expression plasmids can also be
constructed in which the P.sub.ompC1 promoter cassette is replaced
by the P.sub.ompC2 cassette.
It is within the broad scope of the present invention to determine
the stability of these two sets of expression plasmids within CVD
908-htrA to improve and/or optimize the amount of heterologous
antigen produced by a population of live vectors grown for
immunization. Plasmid stability can be estimated by examining
cultures grown both under selection (25 .mu.g/ml neomycin) and
grown without selection by serially passaging cultures using
1:10.sup.6 dilutions as described by Summers and Sheratt Cell,
36:1097, 1984 (incorporated herein by reference), except cultures
can be grown in Super broth with or without 300 mM NaCl rather than
minimal medium. The frequency of plasmid loss can be estimated
using the ratio of colony forming units on selective medium versus
colony forming units on non-selective medium after passaging live
vectors for 0, 25, 50, and 100 generations. The mean fluorescence
of bacteria carrying optimized expression plasmids encoding GFPuv
and grown with or without selection can then be examined using flow
cytometry, and expression of GFPuv from promising constructs by
Western immunoblot analysis using a commercially available
monoclonal antibody specific for GFP (Clontech) can be
confirmed.
Furthermore, it is within the scope of the present invention to
employ within the expression plasmids disclosed herein the proteic
addiction system phd-doc of the temperate bacteriophage P1. As
described above, in proteic addiction systems both the toxin and
antitoxin involve proteins, rather than only RNAs. These proteins
are synthesized from operons in which the gene encoding the
antitoxin is upstream of the gene encoding the toxin.
The phd-doc system encodes two small proteins: the toxic 126 amino
acid Doc protein which causes death on curing by an unknown
mechanism, and the 73 amino acid Phd antitoxin which prevents host
death, presumably by binding to and blocking the action of Doc.
Synthesis of Phd and Doc is translationally coupled, with Phd
synthesis exceeding synthesis of the toxic Doc protein. In
addition, transcription of the operon is autoregulated at the level
of transcription. Although Doc appears to be relatively resistant
to proteolytic attack, Phd is susceptible to cleavage by the ClpXP
serine protease of E. coli.
The PSK mechanism of a plasmid-encoded phd-doc locus is therefore
activated when bacteria spontaneously lose the resident plasmid,
which leads to degradation of the Phd antitoxin and subsequent
activation of the Doc toxin which causes cell death. Therefore,
proper function of the phd-doc system within S. typhi requires the
presence of the ClpXP serine protease; using the published sequence
of the E. coli clpP-clpX operon (GenBank Accession Numbers J05534
and L18867 respectively) PCR of CVD908htrA chromosomal DNA has been
used to demonstrate a product of the expected size presumably
corresponding to the clpP-clpX operon. PCR techniques can also be
employed to construct an Eco RI-Xba I phd-doc cassette for
replacement of the Eco RI-Xba I hok-sok cassette in promising
expression plasmids.
The present invention can also provide an expression plasmid,
expressing a
Plasmid Maintenance System comprising at least one partition locus
and a non-catalytic PSK function, based on trans-complementation of
an otherwise lethal deletion mutation of the CVD 908-htrA
chromosomal ssb gene to improve the observed plasmid-based
expression of heterologous antigens within CVD908-htrA.
The chromosomal ssb locus from CVD 908-htrA can be cloned to
determine the nucleotide sequence of the transcriptional control
region and ssb structural gene, and to construct defined in-frame
deletions to inactivate chromosomally-encoded SSB. The intact ssb
gene can then be inserted into expression plasmids to optimize
expression of the test heterologous antigen, GFPuv.
The non-catalytic PSK function can function as a selectable marker
using the ssb system. Although SSB is an essential protein, it has
no catalytic activity to produce a required product that could be
added to the growth medium allowing ssb mutants to grow. The key to
the success of the proposed chromosomal constructions rests in the
use of two non-leaky conditionally replicative plasmids. One of
these conditional replicons is the temperature-sensitive suicide
vector used f or chromosomal mutagenesis, derived from plB307 and
described above; this suicide vector contains the origin of
replication from pSC101 encoding the temperature-sensitive RepA
protein essential to the function of ori101. The other critical
conditional replicon provides transient expression of SSB protein
in CVD 908-htrA live vectors deleted for ssb, prior to introduction
of expression plasmids carrying the essential ssb gene.
This conditional replicon, designated pCON (conditional replicon)
has recently been constructed, which contains the minimum
functioning origin of replication for oriE1 without the critical
promoter controlling synthesis of RNA I (the promoter controlling
synthesis of the antisense RNA II has not been altered); this RNA I
promoter has been replaced by the Lac repressor-controlled trc
promoter, and the required lacl gene encoding the repressor has
also been included on this plasmid to ensure control of plasmid
replication by the presence or absence of IPTG. IPTG is a chemical
analog of galactose which cannot be cleaved by the enzyme
.beta.-galactosidase. IPTG induces activity of the E. coli lac
operon by binding and inactivating the lac repressor. In the
absence of IPTG, no colony forming units for CVD908-htrA(pCON) were
detected in the presence of carbenicillin selective pressure.
This novel approach significantly extends the range of chromosomal
loci in live vectors now available for mutagenesis.
An expected 3.6 kb fragment for characterization by direct sequence
analysis to define the uvrA-ssb chromosomal locus from CVD908-htrA
has been successfully amplified. Primers homologous to sequences
within the S. typhi soxS gene are currently being used in
combination with primers designed from the partial sequence of the
S. typhi ssb gene to amplify regions of the chromosome flanking the
other side of the CVD908-htrA uvrA-ssb locus. The critical clones
encompassing ssb and flanking regions necessary for the chromosomal
crossovers can then be recovered. Alternatively, a cosmid gene bank
of chromosomal DNA from CVD 908-htrA can be established and the
necessary clone containing ssb and flanking sequences can be
identified using radioactively labelled single-stranded probes
designed from the known partial sequence of the S. typhi Ty2 ssb
gene. Once required clones containing the ssb locus have been
obtained and the sequences for the uvrA-ssb intergenic control
region and ssb are defined, one can proceed with the chromosomal
constructions.
A suicide vector can be constructed in which the sacB-neo cassette
has been inserted upstream of the intact ssb gene, within the
uvrA-ssb intergenic control region, and transcribed toward ssb to
ensure necessary synthesis of SSB. This sacB-neo-ssb cassette can
then be crossed into the chromosome of CVD 908-htrA to associate a
counter-selectable marker with the ssb locus. Merodiploids can be
selected by plating on selective medium containing 25 .mu.g/ml
neomycin and incubating at the non-permissive temperature of
42.degree. C. The required CVD 908-htrA ssb:: sacB-neo strain can
then be selected by plating on selective medium containing 25
.mu.g/ml neomycin and incubating at 30.degree. C.
It is also within the scope of the present invention to prepare a
trc-controlled conditional replicon encoding SSB (designated
pCONssb) and to electroporate this conditional replicon into CVD
908-htrA ssb::sacB-neo; the desired CVD 908-htrA ssb:: sacB -
neo(pCONssb) colonies can be recovered on neomycin-containing
medium supplemented with carbenicillin and 2 mM IPTG to allow for
selection and replication of pCONssb. A further suicide vector can
be constructed in which a defined in-frame deletion mutation which
inactivates ssb can be introduced into a chromosomal fragment
encoding ssb and flanking regions. This .DELTA.ssb suicide vector
can then be electroporated into CVD 908-htrA
ssb::sacB-neo(pCONssb); merodiploids at the chromosomal ssb locus
can be selected for by plating on medium supplemented with 20
.mu.g/ml chloramphenicol, 25 .mu.g/ml neomycin, and 2 mM IPTG, and
incubating at the non-permissive temperature of 42.degree. C.
Resolution of merodiploids and recovery of .DELTA.ssb in the
chromosome can be accomplished by plating at 30.degree. C. on
medium containing 10% sucrose (to counterselect for loss of the
sacB-neo genes), plus carbenicillin and IPTG (to ensure replication
of pCONssb and continued synthesis of SSB), generating CVD 908-htrA
ssb(pCONssb).
An expression plasmid carrying the wildtype ssb gene can then
easily be introduced into CVD 908-htrAssb(pCONssb) to replace
pCONssb, by electroporation and selection of the desired colonies
at 37.degree. C. on non-selective medium without IPTG or
carbenicillin. The ssb cassette can be inserted as an Xba I-Spe I
fragment into all promising expression plasmids to replace the aph
or other selection cassette.
The stability of plasmids carrying the ssb PSK function can then be
determined using the methods described above.
Expression plasmids can be employed which carry mutated ssb genes
analogous to the ssb-1 point mutations identified in E. coli. As
described above, such mutations do not complement chromosomal
lesions in the ssb gene of E. coli unless the mutant SSB-1 proteins
are expressed in high enough amounts from multicopy plasmids. This
approach can therefore enhance the maintenance of proposed
multicopy plasmids containing ori15A or oriE1 origins of
replication.. This is not to say that ssb technology cannot be used
with other systems to further enhance plasmid maintenance. Indeed,
combinations of killing systems reduce loss frequencies by another
10.sup.3 fold versus loss frequencies observed using single killing
systems. However, use of multiple PSK functions in expression
plasmids to optimize synthesis of heterologous antigens must be
balanced against any additional physiological stresses imposed by
multiple killing systems upon the live vector.
A Plasmid Maintenance System based solely on the use of a PSK
function is generally insufficient to guarantee the inheritance of
expression plasmids during division of a host bacterium. Without a
partition function to reduce or eliminate the random inheritance of
expression plasmids, overall expression of heterologous antigens in
a live vector population will drop and reduce immunogenicity. The
random segregation of expression plasmids carrying only a PSK
system can be eliminated by insertion of a partition locus.
Accordingly, in a preferred mode, the present invention comprises
an expression plasmid comprising both a PSK function and at lease
one partitioning function.
The par locus from pSC101 can be used in combination with the oriE1
or ori15A origin of replication, and the parA active partition
locus from pR1 in combination with the oriE1, the ori15A and ori101
origins.
As previously stated, the presence of a PSK function per se does
not affect the frequency at which a given expression plasmid is
inherited; if such a plasmid is unstable, then loss of plasmids
encoding killing functions is expected to produce a decrease in the
overall growth rate of a population of bacteria carrying such
plasmids. When the par locus from pSC101 is incorporated into
expression plasmids that are potentially toxic to host bacteria,
growth rates are improved, presumably due to a reduction of loss
frequencies for resident plasmids. In addition, introduction of the
par locus into medium copy number plasmids such as pBR327 improves
plasmid stability. Insertion of the par locus into high copy number
pUC plasmids can completely stabilize these plasmids in bacteria
serially passaged under non-selective conditions for up to 100
generations. The par locus can therefore improve segregation
frequencies of our expression plasmids in CVD 908-htrA.
Since par does not encode any proteins, use of this locus in
expression plasmids can minimize any metabolic stress on the live
vector due to synthesis of additional foreign proteins. If the par
locus proves insufficient for significantly improving segregation
frequencies, one can consider use of the parA locus from pR1. The
parA active partitioning system is preferred because it naturally
resides within the same plasmid from which the hok-sok locus
originates and is, therefore, compatible with hok-sok in the
expression plasmids.
It is also within the scope of the present invention to provide
expression plasmids which carry a unique Spe I restriction site
(see FIG. 2). Using PCR methods, a Spe I-Nhe I cassette encoding
the par locus from pSC101 can be constructed for insertion into the
Spe I site of plasmids carrying a PSK function. Plasmids in which
par is present in both orientations can be obtained and examined
for any effect of orientation on plasmid maintenance. Since no
proteins are encoded by par, the effects of transcription
originating from other promoters within the expression plasmid are
expected to be minimal. However, this becomes a concern when
attempting to exploit an active partitioning locus since proteins
expressed by such loci are required for proper function of the
locus and must be synthesized at their natural levels. parA-aph Spe
I-Eco RI cassettes in which the aph gene and parA operon are
divergently transcribed and are separated by an Xba I site can be
constructed. This cassette can be used to replace the bla cassettes
of pJN72 and pJN10 (see FIG. 2), and the aph cassette can later be
replaced by the ssb gene or other appropriate locus; origins of
replication can then be replaced by oriE1, ori15A or ori101 Bam
HI-Avr II cassettes from pGEN2 (SEQ. I.D. NO.1), pGEN3 (SEQ. I.D.
NO.2) or pGEN4 (SEQ. I.D. NO.3) respectively (see FIG. 1).
The stability of plasmids carrying Plasmid Maintenance Systems,
comprised of partition and killing functions can then be determined
using the methods described above. These results can be compared to
the stability of plasmids carrying individual partition or killing
cassettes, or no maintenance functions at all.
As mentioned above, another active partitioning locus which
functions in Salmonella is naturally present on the virulence
plasmids of S. typhimurium. Such partition loci are well conserved
among Salmonella virulence plasmids, and when a 3.9 kb restriction
fragment encoding this locus is introduced onto the lower copy
number plasmids containing ori15A, the observed plasmid stability
increases from 34% plasmid-containing cells to 99% plasmid-bearing
cells after 50 generations.
It is within the broad practice of the present invention to insert
this active partition locus into the expression plasmids of the
present invention. The combination of an active partitioning locus
with our proposed ssb technology is expected to significantly
improve plasmid maintenance and overall viability of CVD 908-htrA
carrying these plasmids.
Stx2 is a highly potent toxin strongly implicated in the
development of most HUS cases in the United States. Therefore, to
remove any risk of residual toxicity associated with expression of
a genetically detoxified holotoxin within CVD 908-htrA, the B
subunits of Stx2 can be used as antigens to elicit serum
neutralizing antibodies which block binding of the B pentamer to
Gb.sub.3. It is well established that laboratory strains of E.
coli, capable of plasmid-based expression of wild type Shiga
toxins, are lethal when orally inoculated into mice at high doses.
It has further been demonstrated by ELISA techniques that the
biologically relevant pentameric form of the holotoxin assembles
correctly and is able to bind to its Gb.sub.3 receptor. It is
within the broad practice of the present invention to provide
expression plasmids expressing Stx2.sub.B subunits within CVD
908-htrA resulting in formation of pentamers. Coexpression of an A2
domain can further increase formation of these pentamers.
An operon can be constructed encoding a non-toxic truncated form of
the A subunit in addition to the B subunit of Stx2, designated
stx2.sub.A2B. The truncated A subunit will consist of the natural
leader sequence of Stx2 fused to the A2 domain, and will therefore
not contain the catalytic site responsible for eucaryotic cell
death. This genetically engineered operon encoding Stx2.sub.A2B can
be expressed within CVD 908-htrA from multicopy plasmids which
carry an optimized Plasmid Maintenance System. Expression of
Stx2.sub.A2B pentamers can be compared to expression of Stx2.sub.B
pentamers from an independent cassette encoding only B subunit. It
can be determined if the A2 peptide can enhance proper assembly of
B subunits, preserving neutralizing epitopes within the 5 receptor
binding clefts of the B subunit pentameric ring.
Coexpression of the A2 domain of Stx2 may promote proper assemblage
of .beta.-pentamers for two reasons. First, crystal structure
studies have now demonstrated that the tertiary structure of the B
subunits of the heat-labile enterotoxin LT and Stx1 are remarkably
similar, despite differences in size and a lack of amino acid
sequence identity. It has been further described by Streatfield et
al. that the A2 domain of the closely related cholera and LT
enterotoxins promotes holotoxin assembly and stability in vivo. It
is therefore within the scope of the invention to provide an
expression plasmid wherein expression of the A2 domain can enhance
formation of Stx2 B pentamers. The second reason for coexpression
of the A2 domain relates to the preservation of the conformation of
critical neutralizing epitopes within the binding clefts of the
pentamers. The structure of the B pentamer within the holotoxin (in
which the A subunit is coordinated with the B pentamer via the A2
domain) has more of a 5-fold symmetry than is observed for the
crystal structure of the pentamer alone. It is therefore within the
scope of the invention to provide an expression plasmid wherein
coexpression of A2 will allow formation of pentamers which more
closely resemble naturally occurring pentamers.
Using PCR, an stx2.sub.A2B Bgl II-Nhe I cassette can be constructed
encoding a truncated Stx2 A subunit in which the complete leader
sequence including the terminal serine residue 22 is fused to
residues 262-297 of the mature Stx2 A subunit which will form the
.alpha.-helix that inserts into the central pore of the B pentamer
ring. In order to preserve the natural relative levels of synthesis
of the two subunits, the natural ribosome binding sites upstream of
both the A and B subunits can be preserved.
An stx2.sub.B Bgl II-Nhe I gene cassette can also be constructed
encoding Stx2.sub.B in which an optimal ribosome binding site has
been introduced to promote optimum synthesis of B subunits. Either
of these two cassettes can then be inserted into optimized
expression plasmids which carry Plasmid Maintenance Systems,
replacing gfpuv Bgl II-Nhe I gene cassettes. Expression of
pentamers can be further improved by adjusting the copy number of
the expression plasmids, using the origin of replication cassettes
from either pGEN2 (SEQ. I.D. NO.1), pGEN3 (SEQ. I.D. NO. 2) or
pGEN4 (SEQ. I.D. NO.3), and using alternate promoters such as the
anaerobically activated promoter P.sub.nir15 from pTETnir 15 to
control transcription of these heterologous antigen cassettes.
Residual cytotoxic activity in sonicates of CVD908-htrA expressing
Stx2.sub.A2B and Stx2.sub.B can be detected using the Vero cell
assay. In addition to Vero cells, human renal glomerular
microvascular endothelial (HRMEC) cells can be used to establish
the baseline toxicity of purified Stx2 (necessary for proposed
neutralization assays described below). This assay can be used to
confirm residual toxicity results observed for sonicates assayed
with the standard Vero cell assay.
Generation of immune responses directed against specific immunogens
expressed within attenuated Salmonella can be investigated using
inbred strains of BALB/c mice.
BALB/c mice have been selected because of their serum
immunoglobulin responses to intranasal immunization with live
vaccine carrier strains of Salmonella typhi.
Once CVD908-htrA strains have been established carrying stable
expression plasmids, immunogenicity experiments can be carried out
using constructs expressing either Stx2.sub.A2B or Stx2.sub.B.
BALB/c mice can be randomized to be immunized intranasally with one
of three vaccine strains: 1) CVD908-htrA; 2) CVD908-htrA expressing
Stx2.sub.A2B from unaltered expression plasmids [designated here as
CVD908-htrA(pStx2.sub.A2B)], 3) CVD908-htrA expressing Stx2.sub.A2B
from expression plasmids carrying the optimized Plasmid Maintenance
system [designated CVD908-htrA(pStx2.sub.A2B pm)]. Two intranasal
doses (10.sup.10 CFU in 30 .mu.l) of live vector vaccine can be
administered spaced 28 days apart and sera can be collected before
and 28, 42 and 60 days thereafter to measure titers of Stx2
antitoxin by ELISA, and Stx2 neutralizing antitoxin by the Vero
cell assay. The Vero cell assay neutralizing titers are the
critical endpoints of the experiment.
Based on previous data, it is expected that only 67% of mice given
the parent CVD 908-htrA strain group will attain Shiga toxin
neutralizing antitoxin titers of .gtoreq.1:50 and only 10% of mice
will achieve titers of .gtoreq.1:200. Based on the hypothesis that
in mice immunized with CVD908-htrA(p Stx2.sub.A2B pm) at least 70%
will reach Stx2 antitoxin neutralizing titers of 1:200, inclusion
of 16 mice per group will provide 80% power to detect a significant
difference for each Stx2 versus the control or between the two
expression plasmids with or without the maintenance system
(alpha=0.0167, two-tail test, Bonferroni correction for multiple
comparisons).
After collecting the final serum samples at day 60, mice can be
orally challenged with 10.sup.10 CFU in 100 .mu.l of the E. coli
strain C600(933W) expressing toxigenic Stx2, and observed for 10
days for mortality. Assays for Stx2 neutralizing antitoxin using
the HRMEC assay can also be performed to determine the titer of
Stx2 neutralizing antibodies which block toxicity to the relevant
human tissue postulated to be involved in HUS. These experiments
can be repeated using CVD908-htrA expressing Stx2.sub.B alone to
determine if coexpression of A2 enhances the titer of Stx2
neutralizing antibodies which block binding of the toxin.
It should be noted that one investigator has proposed that Stx 1
binds to Burkitt's lymphoma cells and a subset of tonsillar B
lymphocytes located in germinal centers, causing them to undergo
apoptosis. This proposition has raised some concerns about the
safety of immunizing humans with Stx1 B subunit. It is not known if
this observation applies to Stx2 B subunit as well. Under any
circumstances, in the future, should this concern become
substantiated by clinical observations, it will be possible to
alter Gb.sub.3 binding of either B subunit or mutant Stx2 by
site-directed mutagenesis, as described below.
It is within the scope of the invention to provide an expression
plasmid which expresses within attenuated S. typhi live vector
strains a genetically detoxified and safe Stx2 holotoxin that is
non-toxic yet stimulates neutralizing antitoxin.
It is within the broad scope of the invention to prepare a
detoxified Stx2 holotoxin for expression within CVD908-htrA which
properly assembles into the biologically relevant A1:B5
configuration, preserving neutralizing epitopes within the
catalytic domain (i.e. Stx2.sub.A), as well as preserving the 5
receptor binding clefts of the B subunit (i.e. Stx2.sub.B)
pentameric ring. This genetically engineered mutant Stx2 holotoxin
can be expressed from multicopy plasmids which carry an optimized
Plasmid Maintenance System.
A minimum of two well separated sets of specific point mutations
can be introduced into the open reading frames encoding both the A
and B subunits, in order to remove the possibility of genetic
reversions which restore toxic activity to the holotoxin. The sets
of point mutations can be separated within each reading frame to
remove the possibility of a single rare reversion event reversing
the influence of mutations within a single domain. It is known that
replacement of the phenylalanine residue 30 of Stx1 with alanine
does not alter the three dimensional structure of the resulting
holotoxin. The identical substitution of the analogous residue 29
can therefore be introduced within the B subunit of Stx2, as the
first mutation to detoxify Stx2. This construct can be tested for
immunogenicity and protection in mice. Where protection is
achieved, each additional mutation can be sequentially introduced
and assessed for immunogenicity and protection until all four
mutations are prepared without significantly reducing
immunogenicity of the mutant holotoxin.
It must be emphasized that the original nucleotide sequence
reported for the stx2 operon incorrectly predicted the mature A
subunit to be comprised of 296 residues, rather than 297; the amino
acid coordinates used here for mature Stx2 subunits are based on
GenBank Accession Number X07865, as revised by C. Schmitt, and
referenced in the Addendum of Jackson et al., Journal of
Bacteriology 172:3346, p. 3349, 1990 (incorporated herein by
reference). Based on these coordinates, the following point
mutations can be engineered to create the desired mutant
holotoxins: E167D (i.e. Glu 167.fwdarw.Asp) and W202L (i.e. Trp
202.fwdarw.Leu) to detoxify Stx2a; and Y28F+W29A (i.e. Tyr
28.fwdarw.Phe+Trp29.fwdarw.Ala) and G59D (i.e. Gly59.fwdarw.Asp) to
inactivate the binding sites of Stx2B.
Using overlapping PCR, the following mutations can be introduced
into a Bgl II-Nhe I gene cassette containing mutated stx2
operons:
Restriction Site Sub- Introduced unit Mutation(s) Sequences Changed
(Or Deleted) A E167D (797) - ACA GCA GA{character pullout}
GC{character pullout} TTA - (811) (SEQ ID NO:4) Mlu I W202L (902) -
CTG AAC {character pullout}{character pullout}{character pullout}
GGG CGA - (916) (SEQ ID NO:5) Avr II B Y28F + W29A (1345) - GAA
T{character pullout}C {character pullout}{character pullout}G ACC
AGT - (1359) (SEQ ID NO:6) Eco RI, Nru I (overlapping) G59D (1435)
- GAA TCA G{character pullout}{character pullout} TC{character
pullout} GGA - (1449) (SEQ ID NO:7) Bsp E1 site removed by C
.fwdarw. T substitution. The nucleotide sequence coordinates within
the stx2 operon are listed in parenthesis. Point mutations are
listed in oversized typeface. Restriction sites introduced (or
removed) by these point mutations are denoted by underlined
bases.
The codon usage tables compiled for both E. coli and S. typhi have
been employed in the design of the point mutations. With the
exception of the codon used for the Leu substitution within the A
subunit, none of the codons proposed here is expected to be rare in
S. typhi. Mutated holotoxin cassettes can be inserted into our
optimized expression plasmids, replacing gfpuv cassettes. Lower
copy number plasmids derived from pGEN3 (SEQ. ID. NO.2) and pGEN4
(SEQ. ID. NO.3) can be used for these constructions. Restriction
sites introduced (or deleted) within the stx2 operon can be used to
rapidly identify the desired constructs, and the nucleotide
sequence of promising constructs can be determined to verify the
integrity of each mutation. Promising constructs can be
electroporated into CVD908-htrA, and plasmid maintenance determined
as above. Western immunoblot analysis, using monoclonal antibodies
11E10 (specific for the A subunit of Stx2) and BC5 (specific for
the B subunit of Stx2), can be used to quantify expression of
mutant Stx2 within CVD908-htrA.
Residual cytotoxic activity in sonicates of CVD908-htrA expressing
mutant Stx2 holotoxin can be detected using the Vero cell assay and
the HRMEC assay, as described above.
Once CVD908-htrA strains have been prepared to carry stable
expression plasmids, an initial immunogenicity experiment can be
carried out using constructs expressing Stx2.sub.Y28F+W29A
(designated here as Stx2-1). BALB/c mice can be randomized to be
immunized intranasally with one of three vaccine strains: 1)
CVD908-htrA; 2) CVD908-htrA expressing Stx2-1 from unaltered
expression plasmids [designated here as CVD908-htrA(pStx2-1)], 3)
CVD908-htrA expressing Stx2-1 from expression plasmids carrying the
optimized Plasmid Maintenance system [designated
CVD908-htrA(pStx2-1 pm)]. Two intranasal doses (10.sup.10 CFU in 30
.mu.l) of live vector vaccine can be administered spaced 28 days
apart and sera can be collected before and 28, 42 and 60 days
thereafter to measure titers of Stx2 antitoxin by ELISA, and Stx2
neutralizing antitoxin by the Vero cell assay. The Vero cell assay
neutralizing titers are the critical endpoints of the
experiment.
Based on previous data, only 67% of mice given the parent CVD
908-htrA strain group are expected to attain Shiga toxin
neutralizing antitoxin titers of .gtoreq.1:50 and only 10% of mice
would achieve titers of .gtoreq.1:200. It is expected that in mice
immunized with CVD908-htrA(pStx2-1 pm) at least 70% will reach Stx2
antitoxin neutralizing titers of 1:200. Accordingly, inclusion of
16 mice per group will provide 80% power to detect a significant
difference for each Stx2 versus the control or between the two
expression plasmids with or without the maintenance system
(alpha=0.0167, two-tail test, Bonferroni correction for multiple
comparisons).
After collecting the final serum samples at day 60, mice can be
orally challenged with 10.sup.10 CFU in 100 .mu.l of the E. coli
strain C600(933W), expressing toxigenic Stx2, and observed for 10
days for mortality.
Experiments assaying for Stx2 neutralizing antitoxin using the
HRMEC assay can be used to determine the titer of Stx2 neutralizing
antibodies which block toxicity to the relevant human tissue
postulated to be involved in HUS.
Different promoters can be used to optimize holotoxin expression.
Mutant stx2 operons can be introduced as Bgl II-Bam HI cassettes
into the Bgl II site of a given expression plasmid, creating
stx2(mutant)-gfpuv operons. This will not involve re-engineering of
the cassettes from above since juxtaposed Xba I-Bam HI sites 3' can
be introduced into the cassettes constructed above (i.e. an Xba
I-Bam HI-Nhe I 3'terminus).
The advantage of constructing stx2(mutant)-gfpuv operons is that
transcription levels for mutant stx2 can be monitored by examining
the expression of GFPuv, which is translated from the distal gene
of the polycistronic stx2(mutant)-gfpuv mRNA. Using such a system,
the transcription of mutant stx2 genes can be optimized and can be
monitored indirectly by flow cytometry and confirmed by Western
immunoblot analysis. The gfpuv gene of promising constructs can
then be deleted by digestion with Xba I and Nhe I, and
recircularized constructs can be purified for electroporation into
CVD908-htrA.
7. REFERENCES
The disclosures of the following references are incorporated herein
in their entirety:
Acheson, D. W. K. 1998. Nomenclature of enterotoxins. Lancet
351:1003.
Acheson, D. W. K., M. M. Levine, J. B. Kaper, and G. T. Keusch.
1996. Protective immunity to Shiga-like toxin I following oral
immunization with Shiga-like toxin I B-subunit-producing Vibrio
cholerae CVD 103-HgR. Infection and Immunity 64:355.
Austin, S. J. 1988. Plasmid partition. Plasmid 20:1.
Barry, E. M., O. G. Gomez-Duarte, S. Chatfield, R. Rappuoli, M.
Pizza, G. Losonsky, J. E. Galen, and M. M. Levine. 1996. Expression
and immunogenicity of pertussis toxin S1 subunit-tetanus toxin
fragment C fusions in Salmonella typhi vaccine strain CVD 908.
Infection and Immunity 64:4172.
Bast, D. J., J. L. Brunton, M. A. Karmali, and S. E. Richardson.
1997. Toxicity and immunogenicity of a verotoxin 1 mutant with
reduced globotriaosylceramide receptor binding in rabbits.
Infection and Immunity 65:2019.
Baumler, A. J., J. G. Kusters, I. Stojiljkovic, and F. Heffron.
1994. Salmonella typhimurium loci involved in survival within
macrophages. Infection and Immunity 62:1623.
Blattner, F. R., G. Plunkett III, C. A. Bloch, N. T. Perna, V.
Burland, M. Riley, J. Collado-Vides, J. D. Glasner, C. K. Rode, G.
F. Mayhew, J. Gregor, N. W. Davis, H. A. Kirkpatrick, M. A. Goeden,
D. J. Rose, B. Mau, and Y. Shao. 1997. The complete genome sequence
of Escherichia coli K-12. Science 277.1453.
Blomfield, I. C., V. Vaughn, R. F. Rest, and B. I. Eisenstein.
1991. Allelic exchange in Escherichia coli using the Bacillus
subtilis sacB gene and a temperature-sensitive pSC101 replicon.
Molecular Microbiology 5:1447.
Boe, L. and K. V. Rasmussen. 1996. Suggestions as to quantitative
measurements of plasmid loss. Plasmid 36153.
Boe, L., K. Gerdes, and S. Molin. 1987. Effects of genes exerting
growth inhibition and plasmid stability on plasmid maintenance.
Journal of Bacteriology 169.4646.
Bokman, S. H. and W. W. Ward. 1981. Renaturation of Aequorea
green-fluorescent protein. Biochemical and Biophysical Research
Communications 101:1372.
Bosworth, B. T., J. E. Samuel, H. W. Moon, A. D. O'Brien, V. M.
Gordon, and S. C. Whipp. 1996. Vaccination with genetically
modified Shiga-like toxin IIe prevents edema disease in swine.
Infection and Immunity 64:55.
Bouvier, J., C. Richaud, W. Higgins, O. Bogler, and P. Stragier.
1992. Cloning, characterization, and expression of the dapE gene of
Escherichia coli. Journal of Bacteriology 174:5265.
Boyd, B. and C. A. Lingwood. 1989. Verotoxin receptor glycolipid in
human renal tissue. Nephron 51:207.
Butterton, J. R., E. T. Ryan, D. W. Acheson, and S. B. Calderwood.
1997. Coexpression of the B subunit of Shiga toxin 1 and EaeA from
enterohemorrhagic Escherichia coli in Vibrio cholerae vaccine
strains. Infection and Immunity 65:2127.
Calderwood, S. B., D. W. K. Acheson, G. T. Keusch, T. J. Barrett,
P. M. Griffin, N. A. Strockbine, B. Swaminathan, J. B. Kaper, M. M.
Levine, B. S. Kaplan, H. Karch, A. D. O'Brien, T. G. Obrig, Y.
Takeda, P. I. Tarr, and I. K. Wachsmuth. 1996. Proposed new
nomenclature for SLT (VT) family. ASM News 62.118.
Calderwood, S. B., F. Auclair, A. Donohue-Rolfe, G. T. Keusch, and
J. J. Mekalanos. 1987. Nucleotide sequence of the Shiga-like toxin
genes of Escherichia coli. Proceedings of the National Academy of
Sciences USA 84:4364.
Carlini, L. E., R. D. Porter, U. Curth, and C. Urbanke. 1993.
Viability and preliminary in vivo characterization of site-specific
mutants of Escherichia coli single-stranded DNA-binding protein.
Molecular Microbiology 10.1067.
Carter, P. B. and F. M. Collins. 1974. Growth of typhoid and
paratyphoid bacilli in intravenously infected mice. Infection and
Immunity 10:816.
Cerin, H. and J. Hackett. 1989. Molecular cloning and analysis of
the incompatibility and partition functions of the virulence
plasmid of Salmonella typhimurium. Microbial Pathogenesis 7:85.
Cerin, H. and J. Hackett. 1993. The parVP region of the Salmonella
typhimurium virulence plasmid pSLT contains four loci required for
incompatibility and partition. Plasmid 30:30.
Chalfie, M., Y. Tu, G. Euskirchen, W. W. Ward, and D. C. Prasher.
1994. Green fluorescent protein as a marker for gene expression.
Science 263:802.
Chambers, S. P., S. E. Prior, D. A. Barstow, and N. P. Minton.
1988. The pMTLnic cloning vectors. I. improved pUC polylinker
regions to facilitate the use of sonicated DNA for nucleotide
sequencing. Gene 68:139.
Chase, J. W. and K. R. Williams. 1986. Single-stranded DNA binding
proteins required for DNA replication. Annual Reviews in
Biochemistry 55:103.
Chase, J. W., J. B. Murphy, R. F. Whittier, E. Lorensen, and J. J.
Sninsky. 1983. Amplification of ssb-1 mutant single-stranded
DNA-binding protein in Escherichia coli. Journal of Molecular
Biology 163,164:193.
Chatfield, S., K. Strahan, D. Pickard, I. G. Charles, C. E.
Hormaeche, and G. Dougan. 1992. Evaluation of Salmonella
typhimurium strains harbouring defined mutations in htrA and aroA
in the murine salmonellosis model. Microbial Pathogenesis
12:145.
Clark, C., D. Bast, A. M. Sharp, P. M. St. Hilaire, R. Agha, P. E.
Stein, E. J. Toone, R. J. Read, and J. L. Brunton. 1996.
Phenylalanine 30 plays an important role in receptor binding of
verotoxin-1. Molecular Microbiology 19:891.
Conradi, H. 1903. Ueber losliche,durch aseptische autolyse
erhaltene giftstoffe von ruhr-und Typhusbazillen. Dtsch. Med.
Wochenschr. 29.26.
Covone, M. G., M. Brocchi, E. Palla, W. D. da Silveira, R.
Rappuoli, and C. L. Galeotti. 1998. Levels of expression and
immunogenicity of attenuated Salmonella enterica serovar
typhimurium strains expressing Escherichia coli mutant heat-labile
enterotoxin. Infection and Immunity 66:224.
Dam, M. and K. Gerdes. 1994. Partitioning of plasmid R1: ten direct
repeats flanking the parA promoter constitute a centromere-like
partition site parC, that expresses incompatibility. Journal of
Molecular Biology 236:1289.
Dopf, J. and T. M. Horiagon. 1996. Deletion mapping of the Aequorea
victoria green fluorescent protein. Gene 173:39.
Downes, F. P., T. J. Barrett, J. H. Green, C. H. Aloisio, J. S.
Spika, N. A. Strockbine, and I. K. Wachsmuth. 1988. Affinity
purification and characterization of Shiga-like toxin II and
production of toxin-specific monoclonal antibodies. Infection and
Immunity 56:1926.
Egger, L. A., H. Park, and M. Inouye. 1997. Signal transduction via
the histidyl-aspartyl phosphorelay. Genes to Cells 2:167.
Endo, Y., K. Tsurugi, T. Yutsudo, Y. Takeda, T. Ogasawara, and K.
Igarashi. 1988. Site of action of a Vero toxin (VT2) from
Escherichia coli O157:H7 and of Shiga toxin on eukaryotic
ribosomes: RNA N-glycosidase activity of the toxins. European
Journal of Biochemistry 171:45.
Forrest, B. D., J. T. Labrooy, S. R. Attridge, G. Boehm, L. Beyer,
R. Morona, D. J. C. Shearman, and D. Rowley. 1989. Immunogenicity
of a candidate live oral typhoid/cholera hybrid vaccine in humans.
J. Infect Dis. 159:145.
Fraser, M. E., M. M. Chernaia, Y. V. Kozlov, and M. N. G. James.
1994. Crystal structure of the holotoxin from Shigella dysenteriae
at 2.5 A resolution. Nature Structural Biology 1:59.
Galan, J. E., K. Nakayama, and R. Curtiss III. 1990. Cloning and
characterization of the asd gene of Salmonella typhimurium: use in
stable maintenance of recombinant plasmids in Salmonella vaccine
strains. Gene 94:29.
Galen, J. E. and M. M. Levine. 1995. Improved suicide vectors for
chromosomal mutagenesis in Salmonella typhi. Abstracts of the
Annual Meeting of the American Society of Microbiology
H192:(Abstract)
Galen, J. E. and M. M. Levine. 1996. Further refinements of suicide
vector-mediated chromosomal mutagenesis in Salmonella typhi.
Abstracts of the Annual Meeting of the American Society of
Microbiology H260:(Abstract)
Galen, J. E., O. G. Gomez-Duarte, G. Losonsky, J. L. Halpern, C. S.
Lauderbaugh, S. Kaintuck, M. K. Reymann, and M. M. Levine. 1997. A
murine model of intranasal immunization to assess the
immunogenicity of attenuated Salmonella typhi live vector vaccines
in stimulating serum antibody responses to expressed foreign
antigens. Vaccine 15:700.
Gay, P., D. Le Coq, M. Steinmetz, E. Ferrari, and J. A. Hoch. 1983.
Cloning structural gene sacB, which codes for exoenzyme
levansucrase of Bacillus subtilis: expression of the gene in
Escherichia coli. Journal of Bacteriology 153:1424.
Gerdes, K. and S. Molin. 1986. Partitioning of plasmid R1:
structural and functional analysis of the parA locus. Journal of
Molecular Biology 190:269.
Gerdes, K., A. P. Gultyaev, T. Franch, K. Pedersen, and N. D.
Mikkelsen. 1997. Antisense RNA-regulated programmed cell death.
Annual Reviews in Genetics 31:1.
Gerichter, C. B. 1960. The dissemination of Salmonella typhi, S.
paratyphi A, and S. paratyphi B through the organs of the white
mouse by oral infection. Journal of Hygiene, Cambridge 58:307.
Gerichter, C. B. and D. L. Boros. 1962. Dynamics of infection of
the blood stream and internal organs of white mice with Salmonella
typhi by intraperitoneal injection. Journal of Hygiene, Cambridge
60:311.
Gomez-Duarte, O. G., J. E. Galen, S. N. Chatfield, R. Rappuoli, L.
Eidels, and M. M. Levine. 1995. Expression of fragment C of tetanus
toxin fused to a carboxyl-terminal fragment of diphtheria toxin in
Salmonella typhi CVD 908 vaccine strain. Vaccine 13:1596.
Gonzalez, C., D. M. Hone, F. Noriega, C. O. Tacket, J. R. Davis, G.
Losonsky, J. P. Nataro, S. Hoffman, A. Malik, E. Nardin, M. Sztein,
D. G. Heppner, T. R. Fouts, A. Isibasi, and M. M. Levine. 1994.
Salmonella typhi vaccine strain CVD 908 expressing the
circumsporozoite protein of Plasmodium falciparum: strain
construction and safety and immunogenicity in humans. Journal of
Infectious Diseases 169:927.
Gordon, V. M., S. C. Whipp, H. W. Moon, A. D. O'Brien, and J. E.
Samuel. 1992. An enzymatic mutant of Shiga-like toxin II variant is
a vaccine candidate for edema disease of swine. Infection and
Immunity 60:485.
Gottesman, S., W. P. Clark, V. de Crecy-Lagard, and M. R. Maurizi.
1993. ClpX, an alternative subunit for the ATP-dependent Clp
protease of Escherichia coli. Journal of Biological Chemistry
268:22618.
Green, J. M., B. P. Nichols, and R. G. Matthews. 1996. Folate
biosynthesis, reduction, and polyglutamylation. In Escherichia coli
and Salmonella: Cellular and molecular biology. 2nd ed. F. C.
Neidhardt, R. Curtiss III, J. L. Ingraham, E. C. C. Lin, K. B. Low,
B. Magasanik, W. S. Reznikoff, M. Riley, M. Schaechter and H. E.
Umbarger, eds. ASM Press, Washington, D.C. p. 665.
Griffin, P. M. 1995. Escherichia coli O157:H7 and other
enterohemorrhagic Escherichia coli. In Infections of the
gastrointestinal tract. M. J. Blaser, P. D. Smith, J. I. Ravdin, H.
B. Greenberg and R. L. Guerrant, eds. Raven Press, Ltd, New York,
p. 739.
Gyles, C. L. 1992. Escherichia coli cytotoxins and enterotoxins.
Canadian Journal of Microbiology 38:734.
Heim, R., D. C. Prasher, and R. Y. Tsien. 1994. Wavelength
mutations and posttranscriptional autoxidation of green fluorescent
protein. Proceedings of the National Academy of Sciences USA
91:12501.
Hoiseth, S. K. and B. A. Stocker. 1981. Aromatic-dependent
Salmonella typhimurium are non-virulent and effective as live
vaccines. Nature 291:238.
Hovde, C. J., S. B. Calderwood, J. J. Mekalanos, and R. J. Collier.
1988. Evidence that glutamic acid 167 is an active-site residue of
Shiga-like toxin I. Proceedings of the National Academy of Sciences
USA 85:2568.
Jackson, M. P., E. A. Wadolkowski, D. L. Weinstein, R. K. Holmes,
and A. D. O'Brien. 1990. Functional analysis of the Shiga toxin and
Shiga-like toxin type II variant binding subunits by using
site-directed mutagenesis. Journal of Bacteriology 172:653.
Jackson, M. P., R. J. Neill, A. D. O'Brien, R. K. Holmes, and J. W.
Newland. 1987. Nucleotide sequence analysis and comparison of the
structural genes for Shiga-like toxin I and Shiga-like toxin II
encoded by bacteriophages from Escherichia coli. FEMS Microbiology
Letters 44:109.
Jackson, M. P., R. L. Deresiewicz, and S. B. Calderwood. 1990.
Mutational analysis of the Shiga toxin and Shiga-like toxin II
enzymatic subunits. Journal of Bacteriology 172:3346.
Jarvis, K. G. and J. B. Kaper. 1996. Secretion of extracellular
proteins by enterohemorrhagic Escherichia coli via a putative type
III secretion system. Infection and Immunity 64:4826.
Jarvis, K. G., J. A. Giron, A. E. Jerse, T. K. McDaniel, M. S.
Donnenberg, and J. B. Kaper. 1995. Enteropathogenic Escherichia
coli contains a putative type III secretion system necessary for
the export of proteins involved in attaching and effacing lesion
formation. Proceedings of the National Academy of Sciences USA
92:7996.
Jensen, R. B. and K. Gerdes. 1995. Programmed cell death in
bacteria: proteic plasmid stabilization systems. Molecular
Microbiology 17:205.
Jensen, R. B. and K. Gerdes. 1997. Partitioning of plasmid R1. The
ParM protein exhibits ATPase activity and interacts with the
centromere-like ParR-parC complex. Journal of Molecular Biology
269:505.
Karem, K. L., S. Chatfield, N. Kuklin, and B. T. Rouse. 1995.
Differential induction of carrier antigen-specific immunity by
Salmonella typhimurium live-vaccine strains after single mucosal or
intravenous immunization of BALB/c mice. Infection and Immunity
63.4557.
Karmali, M. A. 1989. Infection by verocytotoxin-producing
Escherichia coli. Clinical Microbiological Reviews 2:15.
Karmali, M. A., M. Petric, C. Lim, P. C. Fleming, and B. T. Steele.
1983. Escherichia coli cytotoxin, haemolytic-uraemic syndrome, and
haemorrhagic colitis. Lancet ii:1299.
Karmali, M. A., M. Petric, C. Lim, P. C. Fleming, G. S. Arbus, and
H. Lior. 1985. The association between idiopathic hemolytic uremic
syndrome and infection by verotoxin-producing Escherichia coli.
Journal of Infectious Diseases 151:775.
Karpman, D., H. Connell, M. Svensson, F. Scheutz, P. Alm, and C.
Svanborg. 1997. The role of lipopolysaccharide and Shiga-like toxin
in a mouse model of Escherichia coli O157:H7 infection. Journal of
Infectious Diseases 175:611.
Keusch, G. T., G. F. Grady, L. J. Mata, and J. McIver. 1972.
Pathogenesis of shigella diarrhea. 1. Enterotoxin production by
Shigella dysenteriae 1. Journal of Clinical Investigation
51:1212.
Killeen, K. P., V. Escuyer, J. J. Mekalanos, and R. J. Collier.
1992. Reversion of recombinant toxoids: mutations in diphtheria
toxin that partially compensate for active-site deletions.
Proceeding of the National Academy of Sciences USA 89:6207.
Kim, J. Y., H. A. Kang, and D. D. Ryu. 1993. Effects of the par
locus on the growth rate and structural stability of recombinant
cells. Biotechnology Progress 9:548.
Konowalchuk, J., J. I. Speirs, and S. Stavric. 1977. Vero response
to a cytotoxin of Escherichia coli. Infection and Immunity
18:775.
Lehnherr, H. and M. B. Yarmolinsky. 1995. Addiction protein Phd of
plasmid prophage P1 is a substrate of the ClpXP serine protease of
Escherichia coli. Proceedings of the National Academy of Sciences
USA 92:3274.
Lehnherr, H., E. Maguin, S. Jafri, and M. B. Yarmolinsky. 1993.
Plasmid addiction genes of bacteriophage P1: doc, which causes cell
death on curing of prophage, and phd, which prevents host death
when prophage is retained. Journal of Molecular Biology
233:414.
Levine, M. M., J. E. Galen, E. M. Barry, F. Noriega, S. Chatfield,
M. Sztein, G.
Dougan, and C. O. Tacket. 1996. Attenuated Salmonella as live oral
vaccines against typhoid fever and as live vectors. Journal of
Biotechnology 44:193.
Lindgren, S. W., J. E. Samuel, C. K. Schmitt, and A. D. O'Brien.
1994. The specific activities of Shiga-like toxin type II (SLT-II)
and SLT-II-related toxins of enterohemorrhagic Escherichia coli
differ when measured by Vero cell cytotoxicity but not by mouse
lethality. Infection and Immunity 62:623.
Lloyd, R. G. and K. B. Low. 1996. Homologous recombination. In
Escherichia coli and Salmonella: Cellular and molecular biology.
2nd ed. F. C. Neidhardt, R. Curtiss III, J. L. Ingraham, E. C. C.
Lin, K. B. Low, B. Magasanik, W. S. Reznikoff, M. Riley, M.
Schaechter and H. E. Umbarger, eds. ASM Press, Washington, D.C. p.
2236.
Lohman, T. M. and M. E. Ferrari. 1994. Escherichia coli
single-stranded DNA-binding protein: multiple DNA-binding modes and
cooperativities. Annual Reviews in Biochemistry 63:527.
Louise, C. B. and T. G. Obrig. 1995. Specific interaction of
Escherichia coli O157:H7-derived Shiga-like toxin II with human
renal endothelial cells. Journal of Infectious Diseases
172.1397.
Love, C. A., P. E. Lilley, and N. E. Dixon. 1996. Stable
high-copy-number bacteriophage lambda promoter vectors for
overproduction of proteins in Escherichia coli. Gene 176:49.
Lynch, A. S. and E. C. C. Lin. 1996. Responses to molecular oxygen.
In Escherichia coli and Salmonella: Cellular and molecular biology.
2nd ed. F. C. Neidhardt, R. Curtiss III, J. L. Ingraham, E. C. C.
Lin, K. B. Low, B. Magasanik, W. S. Reznikoff, M. Riley, M.
Schaechter and H. E. Umbarger, eds. ASM Press, Washington, D.C. p.
1526.
Magnuson, R., H. Lehnherr, G. Mukhopadhyay, and M. B. Yarmolinsky.
1996. Autoregulation of the plasmid addiction operon of
bacteriophage P1. Journal of Biological Chemistry 271:18705.
Mangeney, M., C. A. Lingwood, S. Taga, B. Caillou, T. Tursz, and J.
Wiels. 1993. Apoptosis induced in Burkitt's lymphoma cells via
Gb.sub.3 /CD77, a glycolipid antigen. Cancer Research 53:5314.
Marshall, J., R. Molloy, G. W. J. Moss, J. R. Howe, and T. E.
Hughes. 1995. The jellyfish green fluorescent protein: a new tool
for studying ion channel expression and function. Neuron
14:211.
Matthews, R. G. 1996. One-carbon metabolism. In Escherichia coli
and Salmonella: Cellular and molecular biology. 2nd ed. F. C.
Neidhardt, R. Curtiss III, J. L. Ingraham, E. C. C. Lin, K. B. Low,
B. Magasanik, W. S. Reznikoff, M. Riley, M. Schaechter and H. E.
Umbarger, eds. ASM Press, Washington, D.C. p. 600.
Maurizi, M. R., W. P. Clark, Y. Katayama, S. Rudikoff, J. Pumphrey,
B. Bowers, and S. Gottesman. 1990. Sequence and structure of Clp P,
the proteolytic component of the ATP-dependent Clp protease of
Escherichia coli. Journal of Biological Chemistry 265:12536.
McClelland, M. and R. Wilson. 1998. Sample sequencing of the
Salmonella typhi genome: comparison to the E. coli K-12 genome.
Infection and Immunity.
McDaniel, T. K., K. G. Jarvis, M. S. Donnenberg, and J. B. Kaper.
1995. A genetic locus of enterocyte effacement conserved among
diverse enterobacterial pathogens. Proceedings of the National
Academy of Sciences USA 92:1664.
Melton-Celsa, A. R. and A. D. O'Brien. 1998. The structure,
biology, and relative toxicity for cells and animals of Shiga toxin
family members. In Escherichia coli O157:H7 and other Shiga
toxin-producing E. coli strains. J. B. Kaper and A. D. O'Brien,
eds. ASM Press, Washington, D.C. In press.
Mikkelsen, N. D. and K. Gerdes. 1997. Sok antisense RNA from
plasmid R1 is functionally inactivated by RNaseE and polyadenylated
by poly(A) polymerase I. Molecular Microbiology 26:311.
Moxley, R. A. and D. H. Francis. 1998. Overview of Animal Models.
In Escherichia coli O157:H7 and other Shiga toxin-producing E. coli
strains. J. B. Kaper and A. D. O'Brien, eds. ASM Press, Washington,
D.C. In press.
Muhldorfer, I., J. Hacker, G. T. Keusch, D. W. Acheson, H. Tschape,
A. V. Kane, A. Ritter, T. Olschlager, and A. Donohue-Rolfe. 1996.
Regulation of the Shiga-like toxin II operon in Escherichia coli.
Infection and Immunity 64:495.
Nakayama, K., S. M. Kelley, and R. Curtiss III. 1988. Construction
of an Asd.sup.+ expression-cloning vector: stable maintenance and
high level expression of cloned genes in a Salmonella vaccine
strain. Bio/Technology 6:693.
Nelson, S., S. E. Richardson, C. A. Lingwood, M. Petric, and M. A.
Karmali. 1994. Biological activity of verocytotoxin (VT)2c and
VT1/VT2c chimeras in the rabbit model. In Recent advances in
verocytotoxin-producing Escherichia Coli infections. M. A. Karmali
and A. G. Goglio, eds. Elsevier Science, New York, p. 245.
Nordstrom, K. and S. J. Austin. 1989. Mechanisms that contribute to
the stable segregation of plasmids. Annual Reviews in Genetics
23:37.
Norioka, S., G. Ramakrishnan, K. Ikenaka, and M. Inouye. 1986.
Interaction of a transcriptional activator,OmpR, with reciprocally
osmoregulated genes, ompF and ompC, of Escherichia coli. Journal of
Biological Chemistry 261:17113.
Nyholm, P., G. Magnusson, Z. Zheng, R. Norel, B. Binnington-Boyd,
and C. A. Lingwood. 1996. Two distinct binding sites for
globotriaosyl ceramide on verotoxins: identification by molecular
modelling and confirmation using deoxy analogues and a new
glycolipid receptor for all verotoxins. Chemistry and Biology
3:263.
Nyholm, P., J. L. Brunton, and C. A. Lingwood. 1995. Modelling of
the interaction of verotoxin-1 (VT1) with its glycolipid receptor,
globotriaosylceramide (Gb.sub.3). International Journal of
Biological Macromolecules 17:199.
O'Brien, A. D. 1982. Innate resistance of mice to Salmonella typhi
infection. Infection and Immunity 38:948.
O'Brien, A. D., V. L. Tesh, A. Donohue-Rolfe, M. P. Jackson, S.
Olsnes, K. Sandvig, A. A. Lindberg, and G. T. Keusch. 1992. Shiga
toxin: biochemistry, genetics, mode of action, and role in
pathogenesis. Current Topics in Microbiology and Immunology
180:65.
Olitsky, P. K. and I. J. Kligler. 1920. Toxins and antitoxins of
Bacillus dysenteriae Shiga. Journal of Experimental Medicine
31:19.
Orosz, A., I. Boros, and P. Venetianer. 1991. Analysis of the
complex transcription termination region of the Escherichia coli
rrnB gene. European Journal of Biochemistry 201:653.
Oxer, M. D., C. M. Bentley, J. G. Doyle, T. C. Peakman, I. G.
Charles, and A. J. Makoff. 1991. High level heterologous expression
in E. coli using the anaerobically-activated nirB promoter. Nucleic
Acids Research 19:2889.
Pallen, M. J. and B. W. Wren. 1997. The HtrA family of serine
proteases. Molecular Microbiology 26:209.
Pecota, D. C., C. S. Kim, K. Wu, K. Gerdes, and T. K. Wood. 1997.
Combining the hok/sok, parDE, and pnd postsegregational killer loci
to enhance plasmid stability. Applied and Environmental
Microbiology 63:1917.
Perera, L. P., J. E. Samuel, R. K. Holmes, and A. D. O'Brien. 1991.
Mapping the minimal contiguous gene segment that encodes
functionally active Shiga-like toxin II. Infection and Immunity
59:829.
Perera, L. P., J. E. Samuel, R. K. Holmes, and A. D. O'Brien. 1991.
Identification of three amino acid residues in the B subunit of
Shiga toxin and Shiga-like toxin type II that are essential for
holotoxin activity. Journal of Bacteriology 173:1151.
Pittard, A. J. 1996. Biosynthesis of the aromatic amino acids. In
Escherichia coli and Salmonella: Cellular and molecular biology.
2nd ed. F. C. Neidhardt, R. Curtiss III, J. L. Ingraham, E. C. C.
Lin, K. B. Low, B. Magasanik, W. S. Reznikoff, M. Riley, M.
Schaechter and H. E. Umbarger, eds. ASM Press, Washington, D.C. p.
458.
Polisky, B. 1986. Replication control of the ColE1-type plasmids.
In Maximizing gene expression. W. S. Reznikoff and L. Gold, eds.
Butterworths, Boston, p. 143.
Porter, R. D., S. Black, S. Pannuri, and A. Carlson. 1990. Use of
the Escherichia coli ssb gene to prevent bioreactor takeover by
plasmidless cells. Bio/Technology 8:47.
Pratt, L. A., W. Hsing, K. E. Gibson, and T. J. Silhavy. 1996. From
acids to osmZ: mutiple factors influence synthesis of the OmpF and
OmpC porins in Escherichia coli. Molecular Microbiology 20:911.
Puente, J. L., V. Alvarez-Scherer, G. Gosset, and E. Calva. 1989.
Comparative analysis of the Salmonella typhi and Escherichia coli
ompC genes. Gene 83:197.
Richardson, S. E., T. A. Rotman, V. Jay, C. R. Smith, L. E. Becker,
M. Petric, N. F. Olivieri, and M. A. Karmali. 1992. Experimental
verocytotoxemia in rabbits. Infection and Immunity 60:4154.
Ringquist, S., S. Shinedling, D. Barrick, L. Green, J. Binkley, G.
D. Stormo, and L. Gold. 1992. Translation initiation in Escherichia
coli: sequences within the ribosome-binding site. Molecular
Microbiology 6:1219.
Roberts, M., S. Chatfield, and G. Dougan. 1994. Salmonella as
carriers of heterologous antigens. In Novel delivery systems for
oral vaccines. D. T. O'Hagan, ed. CRC Press, Ann Arbor, p. 27.
Rupp, W. D. 1996. DNA repair mechanisms. In Escherichia coli and
Salmonella: Cellular and molecular biology. 2nd ed. F. C.
Neidhardt, R. Curtiss III, J. L. Ingraham, E. C. C. Lin, K. B. Low,
B. Magasanik, W. S. Reznikoff, M. Riley, M. Schaechter and H. E.
Umbarger, eds. ASM Press, Washington, D.C. p. 2277.
Selzer, G., T. Som, T. Itoh, and J. Tomizawa. 1983. The origin of
replication of plasmid p15A and comparative studies on the
nucleotide sequences around the origin of related plasmids. Cell
32:119.
Siegler, R. L. 1995. The hemolytic uremic syndrome. Pediatric
Nephrology 42:1505.
Siegler, R. L., A. T. Pavia, R. D. Christofferson, and M. K.
Milligan. 1994. A 20-year population-based study of postdiarrheal
hemolytic uremic syndrome in Utah. Pediatrics 94:35.
Sixma, T. K., P. E. Stein, W. G. Hol, and R. J. Read. 1993.
Comparison of the B-pentamers of heat-labile enterotoxin and
verotoxin-1: two structures with remarkable similarity and
dissimilarity. Biochemistry 32:191.
Srinivasan, J., S. A. Tinge, R. Wright, J. C. Herr, and R. Curtiss
III. 1995. Oral immunization with attenuated Salmonella expressing
human sperm antigen induces antibodies in serum and the
reproductive tract. Biology of Reproduction 53:462.
Stein, P. E., A. Boodhoo, G. J. Tyrrell, J. L. Brunton, and R. J.
Read. 1992. Crystal structure of the cell-binding B oligomer of
verotoxin-1 from E. coli. Nature 355:748.
Streatfield, S. J., M. Sandkvist, T. K. Sixma, M. Bagdasarian, W.
G. Hol, and T. R. Hirst. 1992. Intermolecular interactions between
the A and B subunits of heat-labile enterotoxin from Escherichia
coli promote holotoxin assembly and stability in vivo. Proceedings
of the National Academy of Sciences USA 89:12140.
Strockbine, N. A., L. R. M. Marques, J. W. Newland, H. W. Smith, R.
K. Holmes, and A. D. O'Brien. 1986. Two toxin-converting phages
from Escherichia coli O157:H7 strain 933 encode antigenically
distinct toxins with similar biologic activities. Infection and
Immunity 53:135.
Strockbine, N. A., M. P. Jackson, L. M. Sung, R. K. Holmes, and A.
D. O'Brien. 1988. Cloning and sequencing of the genes for Shiga
toxin from Shigella dysenteriae Type 1. Journal of Bacteriology
170:1116.
Summers, D. K. and D. J. Sherratt. 1984. Multimerization of high
copy number plasmids causes instability: ColE1 encodes a
determinant essential for plasmid monomerization and stability.
Cell 36:1097.
Tacket, C. O., D. M. Hone, R. Curtiss III, S. M. Kelly, G.
Losonsky, L. Guers, A. M. Harris, R. Edelman, and M. M. Levine.
1992. Comparison of the safety and immunogenicity of
.DELTA.aroC.DELTA.aroD and .DELTA.cya.DELTA.crp Salmonella typhi
strains in adult volunteers. Infection and Immunity 60:536.
Tacket, C. O., M. Sztein, G. Losonsky, S. S. Wasserman, J. P.
Nataro, R. Edelman, D. Pickard, G. Dougan, S. Chatfield, and M. M.
Levine. 1997. Safety of live oral Salmonella typhi vaccine strains
with deletions in htrA and aroC aroD and immune responses in
humans. Infection and Immunity 65:452.
Tacket, C. O., S. M. Kelley, F. Schodel, G. Losonsky, J. P. Nataro,
R. Edelman, M.
M. Levine, and R. Curtiss III. 1997. Safety and immunogenicity in
humans of an attenuated Salmonella typhi vaccine vector strain
expressing plasmid-encoded hepatitis B antigens stabilized by the
Asd-balanced lethal vector system. Infection and Immunity
65:3381.
Takeda, Y. 1995. Shiga and Siga-like (Vero) toxins. In Bacterial
toxins and virulence factors in disease. J. Moss, B. Iglewski, M.
Vaughan and A. Tu, eds. Marcel Dekker, Inc. New York, p. 313.
Tauxe, R. V. 1998. Public health perspective on immunoprophylactic
strategies for Escherichia coli O157:H7: who or what would we
immunize? In Escherichia coli O157:H7 and other Shiga
toxin-producing E. coli strains. J. B. Kaper and A. D. O'Brien,
eds. ASM Press, Washington, D.C. In press.
Tesh, V. L., J. A. Burris, J. W. Owens, V. M. Gordon, E. A.
Wadolkowski, A. D. O'Brien, and J. E. Samuel. 1993. Comparison of
the relative toxicities of Shiga-like toxins type I and type II for
mice. Infection and Immunity 61:3392.
Thisted, T., A. K. Nielsen, and K. Gerdes. 1994. Mechanism of
post-segregational killing: translation of Hok,SrnB and Pnd mRNAs
of plasmids R1, F and R483 is activated by 3'-end processing. EMBO
Journal 13:1950.
Thisted, T., N. S. Sorensen, and K. Gerdes. 1995. Mechanism of
post-segregational killing: secondary structure analysis of the
entire Hok mRNA from plasmid R1 suggests a fold-back structure that
prevents translation and antisense RNA binding. Journal of
Molecular Biology 247:859.
Thisted, T., N. S. Sorensen, E. G. Wagner, and K. Gerdes. 1994.
Mechanism of post-segregational killing: Sok antisense RNA
interacts with Hok mRNA via its 5'-end single-stranded leader and
competes with the 3'-end of Hok mRNA for binding to the mok
translational initiation region. EMBO Journal 13:1960.
Tinge, S. A. and R. Curtiss III. 1990. Conservation of Salmonella
typhimurium virulence plasmid maintenance regions among Salmonella
serovars as a basis for plasmid curing. Infection and Immunity
58:3084.
Tinge, S. A. and R. Curtiss III. 1990. Isolation of the replication
and partitioning regions of the Salmonella typhimurium virulence
plasmid and stabilization of heterologous replicons. Journal of
Bacteriology 172:5266.
Umbarger, H. E. 1978. Amino acid biosynthesis and its regulation.
Annual Reviews in Biochemistry 47:533.
Valdivia, R. H. and S. Falkow. 1997. Fluorescence-based isolation
of bacterial genes expressed within host cells. Science
277:2007.
Valdivia, R. H., A. E. Hromockyj, D. Monack, L. Ramakrishnan, and
S. Falkow. 1996. Applications for green fluorescent protein (GFP)
in the study of host-pathogen interactions. Gene 173:47.
Vicari, G., A. J. Olitzki, and Z. Olitzki. 1960. The action of the
thermolabile toxin of Shigella dysenteriae on cells cultivated in
vitro. British Journal of Experimental Pathology 41:179.
Wada, K., Y. Wada, F. Ishibashi, T. Gojobori, and T. Ikemura. 1992.
Codon usage tabulated from the GenBank genetic sequence data.
Nucleic Acids Research 20:2111.
Wadolkowski, E. A., L. M. Sung, J. A. Burris, J. E. Samuel, and A.
D. O'Brien. 1990. Acute renal tubular necrosis and death of mice
orally infected with Escherichia coli strains that produce
Shiga-like toxin type II. Infection and Immunity 58:3959.
Wang, S. and T. Hazelrigg. 1994. Implications for bcd mRNA
localization from spatial distribution of exu protein in Drosophila
oogenesis. Nature 369:400.
Wang, Y., Z. Zhang, S. Yang, and R. Wu. 1992. Cloning of par region
and the effect of par region on the stability of pUC9. Chinese
Journal of Biotechnology 8: 107.
Williams, K. R., J. B. Murphy, and J. W. Chase. 1984.
Characterization of the structural and functional defect in the
Escherichia coli single-stranded DNA binding protein encoded by the
ssb-1 mutant gene. Journal of Biological Chemistry 259:11804.
Yamasaki, S., M. Furutani, K. Ito, K. Igarashi, M. Nishibuchi, and
Y. Takeda. 1991. Importance of arginine at postion 170 of the A
subunit of Vero toxin 1 produced by enterohemorrhagic Escherichia
coli for toxin activity. Microbial Pathogenesis 11:1.
Yu, J. and J. B. Kaper. 1992. Cloning and characterization of the
eae gene of enterohaemorrhagic Escherichia coli. Molecular
Microbiology 6:411.
Zalkin, H. and P. Nygaard. 1996. Biosynthesis of purine
nucleotides. In Escherichia coli and Salmonella: Cellular and
molecular biology. 2nd ed. F. C. Neidhardt, J. L. Ingraham, E. C.
C. Lin, K. B. Low, B. Magasanik, W. S. Reznikoff, M. Riley, M.
Schaechter and H. E. Umbarger, eds. ASM Press, Washington, D.C. p.
561.
Zhang, X., Y. Lou, M. Koopman, T. Doggett, K. S. K. Tung, and R.
Curtiss III. 1997. Antibody responses and infertility in mice
following oral immunization with attenuated Salmonella typhimurium
expressing recombinant murine ZP3. Biology of Reproduction
56:33.
Zoja, C., D. Corna, C. Farina, G. Sacchi, C. A. Lingwood, M. P.
Doyle, V. V. Padhye, M. Abbate, and G. Remuzzi. 1992. Verotoxin
glycolipid receptors determine the localization of microangiopathic
process in rabbits given verotoxin-1. Journal of Laboratory and
Clinical Medicine 120:229.
Zurita, M., F. Bolivar, and X. Soberon. 1984. Construction and
characterization of new cloning vehicles. VII. Construction of
plasmid pBR327par, a completely sequenced, stable derivative of
pBR327 containing the par locus of pSC101. Gene 28:119.
SEQUENCE LISTING <100> GENERAL INFORMATION: <160>
NUMBER OF SEQ ID NOS: 7 <200> SEQUENCE CHARACTERISTICS:
<210> SEQ ID NO 1 <211> LENGTH: 4199 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: ()..()
<223> OTHER INFORMATION: pGEN2 nucleotide sequence 1-4199
<400> SEQUENCE: 1 gaattctgtg gtagcacaga ataatgaaaa gtgtgtaaag
aagggtaaaa aaaaccgaat 60 gcgaggcatc cggttgaaat aggggtaaac
agacattcag aaatgaatga cggtaataaa 120 taaagttaat gatgatagcg
ggagttattc tagttgcgag tgaaggtttt gttttgacat 180 tcagtgctgt
caaatactta agaataagtt attgatttta accttgaatt attattgctt 240
gatgttaggt gcttatttcg ccattccgca ataatcttaa aaagttccct tgcatttaca
300 ttttgaaaca tctatagcga taaatgaaac atcttaaaag ttttagtatc
atattcgtgt 360 tggattattc tgcatttttg gggagaatgg acttgccgac
tgattaatga gggttaatca 420 gtatgcagtg gcataaaaaa gcaaataaag
gcatataaca gatcgatctt aaacatccac 480 aggaggatat ctgatgagta
aaggagaaga acttttcact ggagttgtcc caattcttgt 540 tgaattagat
ggtgatgtta atgggcacaa attttctgtc agtggagagg gtgaaggtga 600
tgcaacatac ggaaaactta cccttaaatt tatttgcact actggaaaac tacctgttcc
660 atggccaaca cttgtcacta ctttctctta tggtgttcaa tgcttttccc
gttatccgga 720 tcatatgaaa cggcatgact ttttcaagag tgccatgccc
gaaggttatg tacaggaacg 780 cactatatct ttcaaagatg acgggaacta
caagacgcgt gctgaagtca agtttgaagg 840 tgataccctt gttaatcgta
tcgagttaaa aggtattgat tttaaagaag atggaaacat 900 tctcggacac
aaactcgagt acaactataa ctcacacaat gtatacatca cggcagacaa 960
acaaaagaat ggaatcaaag ctaacttcaa aattcgccac aacattgaag atggatccgt
1020 tcaactagca gaccattatc aacaaaatac tccaattggc gatggccctg
tccttttacc 1080 agacaaccat tacctgtcga cacaatctgc cctttcgaaa
gatcccaacg aaaagcgtga 1140 ccacatggtc cttcttgagt ttgtaactgc
tgctgggatt acacatggca tggatgagct 1200 ctacaaataa tgagctagcc
cgcctaatga gcgggctttt ttttctcggc ctagggccag 1260 caaaaggcca
ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc 1320
cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta
1380 taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt
tccgaccctg 1440 ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa
gcgtggcgct ttctcatagc 1500 tcacgctgta ggtatctcag ttcggtgtag
gtcgttcgct ccaagctggg ctgtgtgcac 1560 gaaccccccg ttcagcccga
ccgctgcgcc ttatccggta actatcgtct tgagtccaac 1620 ccggtaagac
acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg 1680
aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga
1740 aggacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa
aagagttggt 1800 agctcttgat ccggcaaaca aaccaccgct ggtagcggtg
gtttttttgt ttgcaagcag 1860 cagattacgc gcagaaaaaa aggatctcaa
gaagatcctt tgatcttttc tacggggtct 1920 gacgctcagt agatctaaaa
cactaggccc aagagtttgt agaaacgcaa aaaggccatc 1980 cgtcaggatg
gccttctgct taatttgatg cctggcagtt tatggcgggc gtcctgcccg 2040
ccaccctccg ggccgttgct tcgcaacgtt caaatccgct cccggcggat ttgtcctact
2100 caggagagcg ttcaccgaca aacaacagat aaaacgaaag gcccagtctt
tcgactgagc 2160 ctttcgtttt atttgatgcc tggcagttcc ctactctcgc
atggggagac cccacactac 2220 catcggcgct acggcgtttc acttctgagt
tcggcatggg gtcaggtggg accaccgcgc 2280 tactgccgcc aggcaaattc
tgttttatca gaccgcttct gcgttctgat ttaatctgta 2340 tcaggctgaa
aatcttctct catccgccaa aacagccaag ctgggggatc cccgatctta 2400
tcaggtcgag gtggcccggc tccatgcacc gcgacgcaac gcggggaggc agacaaggta
2460 tagggcggcg cctacaatcc atgccaaccc gttccatgtg ctcgccgagg
cggcataaat 2520 cgccgtgacg atcagcggtc cagtgatcga agttaggctg
gtaagagccg cgagcgatcc 2580 ttgaagctgt ccctgatggt cgtcatctac
ctgcctggac agcatggcct gcaacgcggg 2640 catcccgatg ccgccggaag
cgagaagaat cataatgggg aaggccatcc agcctcgcgt 2700 cgcgaacgcc
agcaagacgt agcccagcgc gtcggccgcc atgccggcga taatggcctg 2760
cttctcgccg aaacgtttgg tggcgggacc agtgacgaag gcttgagcga gggcgtgcaa
2820 gattccgaat accgcaagcg acaggccgat catcgtcgcg ctccagcgaa
agcggtcctc 2880 gccgaaaatg acccagagcg ctgccggcac ctgtcctacg
agttgcatga taaagaagac 2940 agtcataagt gcggcgacga tagtcatgcc
ccgcgcccac cggaaggagc tgactgggtt 3000 gaaggctctc aagggcatcg
gtcgacgctc tcccttatgc gactcctgca ttaggaagca 3060 gcccagtagt
aggttgaggc cgttgagcac cgccgccgca aggaatggtg catgcaagga 3120
gatggcgccc aacagtcccc cggccacggg gcctgccacc atacccacgc cgaaacaagc
3180 gctcatgagc ccgaagtggc gagcccgatc ttccccatcg gtgatgtcgg
cgatataggc 3240 gccagcaacc gcacctgtgg cgccggtgat gccggccacg
atgcgtccgg cgtagaggat 3300 ccacaggacg ggtgtggtcg ccatgatcgc
gtagtcgata gtggctccaa gtagcgaagc 3360 gagcaggact gggcggcggc
caaagcggtc ggacagtgct ccgagaacgg gtgcgcatag 3420 aaattgcatc
aacgcatata gcgctagcag cacgccatag tgactggcga tgctgtcgga 3480
atggacgata tcccgcaaga ggcccggcag taccggcata accaagccta tgcctacagc
3540 atccagggtg acggtgccga ggatgacgat gagcgcattg ttagatttca
tttttttttc 3600 ctccttattt tctagacaac atcagcaagg agaaaggggc
taccggcgaa ccagcagccc 3660 ctttataaag gcgcttcagt agtcagacca
gcatcagtcc tgaaaaggcg ggcctgcgcc 3720 cgcctccagg ttgctactta
ccggattcgt aagccatgaa agccgccacc tccctgtgtc 3780 cgtctctgta
acgaatctcg cacagcgatt ttcgtgtcag ataagtgaat atcaacagtg 3840
tgagacacac gatcaacaca caccagacaa gggaacttcg tggtagtttc atggccttct
3900 tctccttgcg caaagcgcgg taagaggcta tcctgatgtg gactagacat
agggatgcct 3960 cgtggtggtt aatgaaaatt aacttactac ggggctatct
tctttctgcc acacaacacg 4020 gcaacaaacc accttcacgt catgaggcag
aaagcctcaa gcgccgggca catcatagcc 4080 catatacctg cacgctgacc
acactcactt tccctgaaaa taatccgctc attcagaccg 4140 ttcacgggaa
atccgtgtga ttgttgccgc atcacgctgc ctcccggagt ttgtctcga 4199
<200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 2
<211> LENGTH: 1200 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: ()..() <223>
OTHER INFORMATION: pGEN3 nucleotide sequence 1201-2397 <400>
SEQUENCE: 2 ctacaaataa tgagctagcc cgcctaatga gcgggctttt ttttctcggc
ctaggagata 60 cttaacaggg aagtgagagg gccgcggcaa agccgttttt
ccataggctc cgcccccctg 120 acaagcatca cgaaatctga cgctcaaatc
agtggtggcg aaacccgaca ggactataaa 180 gataccaggc gtttccccct
ggcggctccc tcgtgcgctc tcctgttcct gcctttcggt 240 ttaccggtgt
cattccgctg ttatggccgc gtttgtctca ttccacgcct gacactcagt 300
tccgggtagg cagttcgctc caagctggac tgtatgcacg aaccccccgt tcagtccgac
360 cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggaaagaca
tgcaaaagca 420 ccactggcag cagccactgg taattgattt agaggagtta
gtcttgaagt catgcgccgg 480 ttaaggctaa actgaaagga caagttttgg
tgactgcgct cctccaagcc agttacctcg 540 gttcaaagag ttggtagctc
agagaacctt cgaaaaaccg ccctgcaagg cggttttttc 600 gttttcagag
caagagatta cgcgcagacc aaaacgatct caagaagatc atcttattaa 660
tcagataaaa tatttctagg atctaaaaca ctaggcccaa gagtttgtag aaacgcaaaa
720 aggccatccg tcaggatggc cttctgctta atttgatgcc tggcagttta
tggcgggcgt 780 cctgcccgcc accctccggg ccgttgcttc gcaacgttca
aatccgctcc cggcggattt 840 gtcctactca ggagagcgtt caccgacaaa
caacagataa aacgaaaggc ccagtctttc 900 gactgagcct ttcgttttat
ttgatgcctg gcagttccct actctcgcat ggggagaccc 960 cacactacca
tcggcgctac ggcgtttcac ttctgagttc ggcatggggt caggtgggac 1020
caccgcgcta ctgccgccag gcaaattctg ttttatcaga ccgcttctgc gttctgattt
1080 aatctgtatc aggctgaaaa tcttctctca tccgccaaaa cagccaagct
gggggatccc 1140 cgatcttatc aggtcgaggt ggcccggctc catgcaccgc
gacgcaacgc ggggaggcag 1200 <200> SEQUENCE CHARACTERISTICS:
<210> SEQ ID NO 3 <211> LENGTH: 2650 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: ()..()
<223> OTHER INFORMATION: pGEN4 nucleotide sequence 1201-3847
<400> SEQUENCE: 3 ctacaaataa tgagctagcc cgcctaatga gcgggctttt
ttttctcggc ctaggtttca 60 cctgttctat taggtgttac atgctgttca
tctgttacat tgtcgatctg ttcatggtga 120 acagctttaa atgcaccaaa
aactcgtaaa agctctgatg tatctatctt ttttacaccg 180 ttttcatctg
tgcatatgga cagttttccc tttgatatct aacggtgaac agttgttcta 240
cttttgtttg ttagtcttga tgcttcactg atagatacaa gagccataag aacctcagat
300 ccttccgtat ttagccagta tgttctctag tgtggttcgt tgtttttgcg
tgagccatga 360 gaacgaacca ttgagatcat gcttactttg catgtcactc
aaaaattttg cctcaaaact 420 ggtgagctga atttttgcag ttaaagcatc
gtgtagtgtt tttcttagtc cgttacgtag 480 gtaggaatct gatgtaatgg
ttgttggtat tttgtcacca ttcattttta tctggttgtt 540 ctcaagttcg
gttacgagat ccatttgtct atctagttca acttggaaaa tcaacgtatc 600
agtcgggcgg cctcgcttat caaccaccaa tttcatattg ctgtaagtgt ttaaatcttt
660 acttattggt ttcaaaaccc attggttaag ccttttaaac tcatggtagt
tattttcaag 720 cattaacatg aacttaaatt catcaaggct aatctctata
tttgccttgt gagttttctt 780 ttgtgttagt tcttttaata accactcata
aatcctcata gagtatttgt tttcaaaaga 840 cttaacatgt tccagattat
attttatgaa tttttttaac tggaaaagat aaggcaatat 900 ctcttcacta
aaaactaatt ctaatttttc gcttgagaac ttggcatagt ttgtccactg 960
gaaaatctca aagcctttaa ccaaaggatt cctgatttcc acagttctcg tcatcagctc
1020 tctggttgct ttagctaata caccataagc attttcccta ctgatgttca
tcatctgagc 1080 gtattggtta taagtgaacg ataccgtccg ttctttcctt
gtagggtttt caatcgtggg 1140 gttgagtagt gccacacagc ataaaattag
cttggtttca tgctccgtta agtcatagcg 1200 actaatcgct agttcatttg
ctttgaaaac aactaattca gacatacatc tcaattggtc 1260 taggtgattt
taatcactat accaattgag atgggctagt caatgataat tactagtcct 1320
tttcctttga gttgtgggta tctgtaaatt ctgctagacc tttgctggaa aacttgtaaa
1380 ttctgctaga ccctctgtaa attccgctag acctttgtgt gttttttttg
tttatattca 1440 agtggttata atttatagaa taaagaaaga ataaaaaaag
ataaaaagaa tagatcccag 1500 ccctgtgtat aactcactac tttagtcagt
tccgcagtat tacaaaagga tgtcgcaaac 1560 gctgtttgct cctctacaaa
acagacctta aaaccctaaa ggcttaagta gcaccctcgc 1620 aagctcgggc
aaatcgctga atattccttt tgtctccgac catcaggcac ctgagtcgct 1680
gtctttttcg tgacattcag ttcgctgcgc tcacggctct ggcagtgaat gggggtaaat
1740 ggcactacag gcgcctttta tggattcatg caaggaaact acccataata
caagaaaagc 1800 ccgtcacggg cttctcaggg cgttttatgg cgggtctgct
atgtggtgct atctgacttt 1860 ttgctgttca gcagttcctg ccctctgatt
ttccagtctg accacttcgg attatcccgt 1920 gacaggtcat tcagactggc
taatgcaccc agtaaggcag cggtatcatc aacaggctta 1980 cccgtcttac
tgtcaaccgg atctaaaaca ctaggcccaa gagtttgtag aaacgcaaaa 2040
aggccatccg tcaggatggc cttctgctta atttgatgcc tggcagttta tggcgggcgt
2100 cctgcccgcc accctccggg ccgttgcttc gcaacgttca aatccgctcc
cggcggattt 2160 gtcctactca ggagagcgtt caccgacaaa caacagataa
aacgaaaggc ccagtctttc 2220 gactgagcct ttcgttttat ttgatgcctg
gcagttccct actctcgcat ggggagaccc 2280 cacactacca tcggcgctac
ggcgtttcac ttctgagttc ggcatggggt caggtgggac 2340 caccgcgcta
ctgccgccag gcaaattctg ttttatcaga ccgcttctgc gttctgattt 2400
aatctgtatc aggctgaaaa tcttctctca tccgccaaaa cagccaagct gggggatccc
2460 cgatcttatc aggtcgaggt ggcccggctc catgcaccgc gacgcaacgc
ggggaggcag 2520 acaaggtata gggcggcgcc tacaatccat gccaacccgt
tccatgtgct cgccgaggcg 2580 gcataaatcg ccgtgacgat cagcggtcca
gtgatcgaag ttaggctggt aagagccgcg 2640 agcgatcctt 2650 <200>
SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 4 <211>
LENGTH: 15 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <221> NAME/KEY: misc_feature
<222> LOCATION: ()..() <223> OTHER INFORMATION: mutated
Shiga toxin segment <400> SEQUENCE: 4 acagcagacg cgtta 15
<200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 5
<211> LENGTH: 15 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: ()..() <223> OTHER
INFORMATION: mutated Shiga toxin segment <400> SEQUENCE: 5
ctgaacctag ggcga 15 <200> SEQUENCE CHARACTERISTICS:
<210> SEQ ID NO 6 <211> LENGTH: 15 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: ()..()
<223> OTHER INFORMATION: mutated Shiga toxin segment
<400> SEQUENCE: 6 gaattcgcga ccagt 15 <200> SEQUENCE
CHARACTERISTICS: <210> SEQ ID NO 7 <211> LENGTH: 15
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: ()..() <223> OTHER INFORMATION: mutated Shiga toxin
segment <400> SEQUENCE: 7 gaatcagatt ctgga 15
* * * * *